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AQUEOUS REMEDIATION OF 4,4'-DICHLOROBIPHENYL
BY F ENTON’S REAGENT: A STUDY OF OXIDATIVE
DEGRADATION, BYPRODUCT PRODUCTION, AND

TOXICOLOGICAL EFFECT

presented by

Andrea Yuki Satoh

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5/08 K:IProVAccS-Pres/ClRC/DateDueindd

AQUEOUS REMEDIATION OF 4,4’-DICHLOROBIPHENYL BY FENTON’S
REAGENT: A STUDY OF OXIDATIVE DEGRADATION, BYPRODUCT
PRODUCTION, AND TOXICOLOGICAL EFFECT
By

Andrea Yuki Satoh

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree Of
DOCTOR OF PHILOSOPHY

Environmental Engineering

2008

ABSTRACT
AQUEOUS REMEDIATION OF 4,4'-DICHLOROBIPHENYL BY FENTON’S
REAGENT: A STUDY OF OXIDATIVE DEGRADATION, BYPRODUCT
PRODUCTION, AND TOXICOLOGICAL EFFECT
By
Andrea Yuki Satoh
2-biphenylol (ZBP), 3-biphenylol (3BP), 2,2’-biphenyldiol (2,2'BP), 3,3'-

biphenyldiol (3,3'BP), 3-chlorO-2-biphenylol (3C2BP), and 4,4’-dichloro-3-biphenylol
(4,4'DC3BP), considered to be representative of potential byproducts Of Fenton’s
remediation of polychlorinated biphenyls (PCBs), were studied for the correlation
between their chemical/ structural properties and Observed epigenetic toxicity and
cytotoxicity. The scrape-loading/dye transfer (SL/DT) technique was performed to
determine the in vitro modulation of gap junctional intercellular communication (GJIC)
in a normal rat liver epithelial cell line as a measure of the epi genetic toxicity.
Cytotoxicity was determined using the neutral red uptake assay. Only 3,3'BP and
4,4'DC3BP induced cytotoxicity within a dose range of 0 to 300 M. 4,4'DC3BP was
most inhibitory to GJIC at the lowest dose. 3CZBP was least inhibitory to GJIC.
Although cells were capable of complete recovery of GJIC after removal of each of the
chemicals, only with 2,2'BP and 4,4'DC3BP did the cells demonstrate partial recovery
without removal of the chemical. In view of the cytotoxicity and GJIC inhibitory effects
observed for 4,4’DC3BP, the PCB congener 4,4'-dichlorobiphenyl (4,4’DCBP) was
selected for F enton’s reagent remediation studies. For toxicology studies performed for

4,4'DCBP, over a dose range of O to 260.87 M, very slight to no inhibition Of GJIC was

observed for incubation times of 30 minutes, 2 hours, 6 hours, and 24 hours.

The methylene blue (MB) dye test was developed to qualitatively indicate the
presence of hydroxyl radicals through an immediate, distinct bleaching of the MB dye on
a paper test strip. When applied to F enton’s remediation, MB dye tests were capable of
indicating the formation of hydroxyl radicals during the Fenton’s reaction and indicating
the completion of quenching. The presence of hydroxyl radicals was verified by benzoic
acid chemical probe hydroxyl radical detection methods using thin layer chromatography
(TLC) and spectrophotometric wavelength scans.

Fenton’s remediation of 4,4'DCBP in 50/50 Milli-Q water/acetonitrile was
performed, followed by an examination of the toxicity of the final F enton’s remediation
solution and disappearance of 4,4'DCBP as a result of F enton’s remediation. The
Fenton’s reaction conditions were pH 3.0, temperature 23.0 °C, Fe2+szOz molar ratio
1:20, initial Fe2+ concentration 0.15 mM, and initial H202 concentration 3 mM. Partial
inhibition of GJIC occurred for test volumes greater than 40 uL (30 minute incubation),
and a maximum level of inhibition was attained at 60 uL (FOC = 0.75 i 0.03). For the
time-response GJIC bioassay (60 uL test volume), a maximum level of inhibition was
attained at 30 minutes (F OC = 0.78 i 0.02), followed by complete recovery of GJIC
without removal of the chemical by 240 minutes. Although the final F enton’s
remediation solution was determined to be more toxic than the parent PCB, and the
inhibitory effects were similar to those observed for 4,4'DC3BP, the byproducts
responsible for the toxicity observed could not be ascertained by the detection methods
employed. Only 27.22 i 1.32% of the 4,4'DCBP remained by 15 minutes of remediation

and no further decrease occurred through 60 minutes.

Copyright by
ANDREA YUKI SATOH
2008

I dedicate this dissertation to my family, whose unending support has encouraged me on
this long journey, and to my Grandpa, Dr. John S. Evans, who taught me to believe in my
dreams.

ACKNOWLEDGNIENTS

My thanks and appreciation to each member of my committee for their
contributions towards the completion of this research and dissertation. I sincerely
appreciate the continual help, support, and encouragement that Dr. James E. Trosko has
provided me. His willingness to always make time to listen to me has been a constant
source of reassurance. No matter what questions I might have had or problems I might
have encountered, Dr. Trosko always seemed to have an answer or solution that would
restore the balance to both my academic and personal life. I am especially grateful to Dr.
Trosko for enhancing my background in environmental engineering with a knowledge of
toxicology, gap junctional intercellular communication, and cancer research. I appreciate
the contributions of support and assistance from Dr. Susan J. Masten throughout the
completion of my degree, despite a separation of geographical distance. I am most
appreciative of Dr. Bruce Dale for being a committee member and offering me assistance
during critical last minute changes. The Department of Chemical Engineering and
Materials Science is very fortunate to have Dr. Dale as a faculty member. I thank Dr.
Patricia Ganey for being a member of my committee.

I am grateful to several individuals for academic and technical assistance
throughout my research. I thank Dr. Chia-Cheng Chang for his generosity in the use of
laboratory space, equipment, and supplies, as well as emotional support and spontaneous
advising. His friendship has been a valuable resource. I am thankful to Yanlyang Pan for
his technical assistance in performing gas chromatography for my research. I will always

be grateful to Dr. Karen Klomparens, Dean of the Graduate School, for her

vi

encouragement and support to continue my graduate studies despite my life-threatening
illness.

I acknowledge the following sources of funding: NIEHS-Superfund Grant P42
E80491 1, Jerry N. McCowan Endowed Fellowship, Summer Retention Fellowship, and
Dissertation Completion Fellowship (Michigan State University Graduate School).

I thank my family and friends for their encouragement, patience, and support
throughout these many years of doctoral research and during the dissertation writing
process. A special thanks to Dr. Ronald Crafion for his extensive emotional support,
advice, and focus. Finally, I acknowledge my fi'iends and loved ones who could not
complete this journey with me. Although I miss them all greatly, my memories of each

of them have inspired me.

vii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................... xii
LIST OF FIGURES .............................................................................. xiii
ABBREVIATIONS .............................................................................. xxi
CHAPTER 1: INTRODUCTION

1.1 Background and Environmental Significance ........................................... l
1.2 Remediation of Polychlorinated Biphenyls Using Fenton’s Reagent ................ 5
1.3 Toxicity of Hydroxylated Polychlorinated Biphenyls (OH-PCBs) ................... 8
1.4 Gap Junctional Intercellular Communication (GJIC) as a Marker of Toxicity. . . 9
1.5 Hypotheses and Objectives ................................................................ 11
1.6 Organization of Dissertation ............................................................... 13
1.7 References .................................................................................... 17

CHAPTER 2: EPIGENETIC TOXICITY OF HYDROXYLATED BIPHENYLS
AND HYDROXYLATED POLYCHLORINATED BIPl-IENYLS AS POTENTIAL

REMEDIATION BYPRODUCTS

2.1 Introduction .................................................................................. 20
2.2 Experimental Section ....................................................................... 22
2.2.1 Chemicals ............................................................................. 22
2.2.2 Methods ............................................................................... 24
2.2.2.1 Toxicological Evaluation ................................................. 24
2.2.2.2 Cell Culture Techniques ................................................... 25
2.2.2.3 In Vitro Bioassay for GJIC ................................................ 26
{ 2.2.2.4 In Vitro Bioassay for Cytotoxicity ....................................... 28
2.3 Results and Discussion ..................................................................... 31
2.3.1 30-Minute Cytotoxicity Assay ..................................................... 31
2.3.2 Dose-Response Bioassay ............................................................ 34
2.3.3 24-Hour Cytotoxicity Assay ........................................................ 40
2.3.4 Time-Response Bioassay ........................................................... 42
2.3.5 Time of Recovery Bioassay ........................................................ 46

2.3.6 Estimation of OctanOl/Water Partition Coefficients and Solubility in
Water .................................................................................. 50
2.3.7 Structure-Toxicity Relationships ................................................... 52
2.4 Conclusions .................................................................................. 55
2.5 References .................................................................................... 57

viii

CHAPTER 3: TOXICITY OF PARENT POLYCHLORINATED BIPHENYL

(4,4'-DICHLOROBIPHENYL)

3.1 Introduction .................................................................................. 61
3.2 Experimental Section ....................................................................... 62
3.2.1 Chemicals ............................................................................. 62
3.2.2 Methods ............................................................................... 63
3.2.2.1 Cell Culture Techniques ................................................... 63
3.2.2.2 Solvent Evaluation for GJIC Bioassay .................................. 63

3. 2. 2. 3 PCB “Transport” Using DMSO/BSA/PBS Solvent for GJIC
Bioassay ..................................................................... 64
3. 2. 2. 4 Vehicle Tolerance Test for DMSO/BSA/PBS ......................... 65
3.2.2.5 Dose-Response Bioassay ................................................. 67
3.3 Results and Discussion ..................................................................... 70
3.3.1 Solvent Evaluation for GJIC Bioassay ............................................ 70
3.3.2 Vehicle Tolerance Test for DMSO/BSA/PBS ................................... 71
3.3.3 Dose-Response Bioassay ............................................................ 71
3.4 Conclusions .................................................................................. 76
3.5 References .................................................................................... 78

CHAPTER 4: METHYLENE BLUE DYE TEST FOR RAPID QUALITATIVE
DETECTION OF HYDROXYL RADICALS FORMED IN A FENTON’S
REACTION AQUEOUS SOLUTION

4.1 Introduction .................................................................................. 80
4.2 Experimental Section ....................................................................... 82
4.2.1 Chemicals ............................................................................. 82
4.2.2 Methods ............................................................................... 83
4.2.2.1 Methylene Blue Dye Test ................................................. 83
4.2.2.2 Fenton’s Reaction in Milli-Q Water ..................................... 85
4.2.2.3 Hydroxyl Radical Detection by Benzoic Acid ......................... 86
4.2.2.4 Thin-Layer Chromatography (TLC) .................................... 87
4.2.2.5 Spectrophotometric Wavelength Scans ................................. 89
4.3 Results and Discussion ..................................................................... 89
4.3.1 F enton’s Reaction in Milli-Q Water (N o Benzoic Acid Addition) ............ 89
4.3.2 Benzoic Acid in an Unquenched Fenton’s Reaction Mixture .................. 92
4.3.3 TLC of an Unquenched Fenton’s Reaction Mixture without BA Addition. 103
4.3.4 Benzoic Acid in a Quenched Fenton’s Reaction Mixture ...................... 103
4.3.5 Methylene Blue Dye Test Strip Studies: Age, 3% H202, and Influence
of pH ................................................................................... 105
4.4 Conclusions .................................................................................. 109
4.5 References .................................................................................... 11 1
CHAPTER 5: TOXICOLOGY STUDIES OF FENTON’S REMEDIATION IN
MILLI-Q WATER
5.1 Introduction .................................................................................. 113
5.2 Experimental Section ....................................................................... 114
5.2.1 Chemicals ............................................................................. 114

ix

5.2.2 Methods ............................................................................... 115

5.2.2.1 Fenton’s Remediation in Milli-Q Water ................................ 115

5.2.2.2 Cell Culture Techniques ................................................... 117

5.2.2.3 In Vitro Bioassay for GJIC ................................................ 118

5.3 Results and Discussion ..................................................................... 121
5.3.1 Fenton’s Remediation in Milli-Q Water .......................................... 121
5.3.2 In Vitro Bioassay for GJIC ......................................................... 128

5.4 Conclusions .................................................................................. 132
5.5 References .................................................................................... 133

CHAPTER 6: TOXICOLOGY STUDIES OF F ENTON’S REMEDIATION OF
POTENTIAL SOLVENTS FOR 4,4'-DICHLOROBIPHENYL

6.1 Introduction .................................................................................. 135
6.2 Experimental Section ....................................................................... 136
6.2.1 Chemicals ............................................................................. 136
6.2.2 Methods ............................................................................... 137
6.2.2.1 Preliminary Methylene Blue Dye Tests ................................. 137

6.2.2.2 Fenton’s Remediation of 80/20 and 50/50 Milli-Q
Water/Acetonitrile ......................................................... 137
6.2.2.3 Cell Culture Techniques ................................................... 141
6.2.2.4 In Vitro Bioassay for GJIC ................................................ 141
6.3 Results and Discussion ..................................................................... 143
6.3.1 Preliminary Methylene Blue Dye Tests .......................................... 143
6.3.2 F enton’s Remediation of 80/20 and 50/ 50 Milli-Q Water/Acetonitrile ...... 145
6.3.3 In Vitro Bioassay for GJIC ......................................................... 155
6.4 Conclusions .................................................................................. 159
6.5 References .................................................................................... 161

CHAPTER 7: FENTON’S REMEDIATION OF 4,4'-DICHLOROBIPHENYL
[N 50/50 MILLI-Q WATER/ACETONITRILE AND TOXICITY OF
REMEDIATION MIXTURE

7.1 Introduction .................................................................................. 162

7.2 Experimental Section ....................................................................... 163

7.2.1 Chemicals ............................................................................. 163

7.2.2 Methods ............................................................................... 164
7.2.2.1 Fenton’s Remediation of 4,4'-Dichlorobiphenyl in 50/50

Milli-Q HzO/ACN ......................................................... 164

7.2.2.2 Cell Culture Techniques ................................................... 168

7.2.2.3 In Vitro Bioassay for GJIC ................................................ 169

7.3 Results and Discussion ..................................................................... 173

7.3.1 Fenton’s Remediation of 4,4'-Dichlorobiphenyl in 50/50

Milli-Q HZO/ACN ................................................................... 173

7.3.2 Dose-Response Bioassay ............................................................ 179

7.3.3 Time-Response Bioassay ........................................................... 179

7.4 Conclusions .................................................................................. 185

7.5 References .................................................................................... 188

CHAPTER 8: TIME COURSE STUDIES: FENTON’S REMEDIATION OF
4,4'-DICHLOROBIPHENYL IN 50/50 MILLI-Q WATER/ACETONITRILE
AND DISAPPEARANCE OF PARENT PCB

8.1 Introduction .................................................................................. 190
8.2 Experimental Section ....................................................................... 191
8.2.1 Chemicals ............................................................................. 191
8.2.2 Methods ............................................................................... 192
8.2.2.1 Gas Chromatography/Electron Capture Detector (GC/ECD). . . . . 192
8.2.2.2 Calibration Curve .......................................................... 193
8.2.2.3 Percent Efficiency of Recovery by Isooctane Extraction ............. 193
8.2.2.4 Fenton’s Remediation with Varying Reaction Times ................. 195
8.2.2.5 Isooctane Extraction Procedure .......................................... 200
8.2.2.6 Fenton’s Remediation Solution Concentration by
Back-Calculation ........................................................... 201
8.2.2.7 Retention Times for Chlorinated Potential Remediation
Byproducts .................................................................. 203
8.3 Results and Discussion ..................................................................... 204
8.3.1 Retention Times for Chlorinated Potential Remediation Byproducts. . . . . 204
8.3.2 Percent Efficiency of Recovery by Isooctane Extraction ....................... 204
8.3.3 Fenton’s Remediation of 4,4'-Dichlorobiphenyl and Extraction .............. 217
8.3.4 GC/ECD Analysis and Disappearance of Parent PCB .......................... 225
8.4 Conclusions .................................................................................. 245
8.5 References .................................................................................... 247
CHAPTER 9: SUMMARY AND RECOMMENDATIONS
9.1 Summary ..................................................................................... 249
9.2 Recommendations ........................................................................... 254

xi

LIST OF TABLES

Table 2.1 24 Hour Cytotoxicity at the Doses Used for the Time-Response
Bioassays ........................................................................... 41

Table 2.2 Estimated OctanoI/Water Partition Coefficients and Water
Solubility ........................................................................... 5 1

Table 4.1 Potential Eluting Solvents Investigated for TLC Plate Development. . ....88
Table 4.2 Retention Factor (Rf) Results for Benzoic Acid Chemical Probe in
an Unquenched (Plates A-F) and Quenched (Plates G-J) F enton’s
Reaction Mixture .................................................................. 97

Table 5.1 300 uL GJIC Assay Results for 30 Minutes and 2 Hour Incubation
Times .............................................................................. 128

xii

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

LIST OF FIGURES

Images in this dissertation are presented in color.

Chemical structures of the six chemicals selected as potential
byproducts of remediation of PCBs with Fenton’s reagent and
evaluated for toxicity ............................................................. 23

Cytotoxicity results using the neutral red uptake assay for each of the

six selected potential remediation byproducts. Cell cultures were

exposed to the chemicals for 30 minutes. Each chemical was tested

for a dose range of 0 to 300 M. Each data point is representative of

the results for a set of chemically treated triplicates reported as an

average F OC i standard deviation determined at the 95% confidence
interval .............................................................................. 33

Dose—response results for each of the six selected potential

remediation byproducts. Cell cultures were exposed to the chemicals

for 30 minutes. Each chemical was tested for a dose range of 0 to 300
uM. Each data point is representative of the results for a set of
chemically treated triplicates reported as an average FOC i standard
deviation determined at the 95% confidence interval ........................ 36

Phase contrast (visible light) and UV epifluorescent
photomicrographs at 200x magnification comparing 0 and 250 uM
doses of 4,4'DC3BP. A treatment of 0 uM 4,4'DC3BP for 30 minutes
was a vehicle control treatment with 25 uL of ACN for 30 minutes.
(A) Phase contrast and (C) UV epifluorescent photomicrographs of
cell cultures treated with 0 uM 4,4'DC3BP for 30 minutes indicate a
confluent layer of healthy cells with complete communication.

(B) Phase contrast and (D) UV epifluorescent photomicrographs of
cell cultures treated with 250 uM 4,4'DC3BP for 30 minutes indicate
detachment of cells from the cell monolayer and a fluorescing of the
remaining cells ..................................................................... 39

Time-response results for each of the six selected potential

remediation byproducts. The legend in the figure indicates the dose

used for each of the chemicals. Time of exposure varied from

0 minutes to 1440 minutes (24 hours). Each data point is

representative of the results for a set of chemically treated triplicates
reported as an average FOC :t standard deviation determined at the

95% confidence interval. Data points after the break correspond to

120 minutes, 240 minutes, 360 minutes, 480 minutes, 600 minutes,

720 minutes, and 1440 minutes of chemical exposure, respectively ....... 44

xiii

Figure 2.6 Time of recovery results for five of the six selected potential
remediation byproducts. Since no significant level of inhibition
was observed in the time-response results for 3C2BP, no time of
recovery experiment was performed for this chemical. The legend
in the figure indicates the dose and incubation time selected for the
time of recovery experiment for each chemical. Each data point is
representative of the results for a set of chemically treated triplicates
reported as an average FOC i standard deviation determined at the
95% confidence interval. Data points after the break correspond to
60 minutes, 120 minutes, 240 minutes, 360 minutes, 480 minutes,
and 600 minutes of recovery, respectively .................................... 48

Figure 3.1 Vehicle tolerance test GJIC bioassay results with a 30 minute
incubation time for a volume range of 0 to 350 uL of
DMSO/BSA/PBS. Each data point is representative of the results
for a set of DMSO/BSA/PBS treated triplicates reported as an
average FOC i standard deviation determined at the 95%
confidence interval ................................................................ 72

Figure 3.2 Dose-response bioassay results for 4,4'-dichlorobiphenyl
(DMSO/BSA/PBS solvent) with 30 minutes of incubation time for
a dose range of O to 260.87 uM. Each data point is representative
of the results for a set of chemically treated triplicates reported as
an average FOC i- standard deviation determined at the 95%
confidence interval ................................................................ 73

Figure 3.3 Dose-response bioassay results for 4,4'-dichlorobiphenyl
(DMSO/BSA/PBS solvent) with 2 hours of incubation time for
a dose range of 0 to 260.87 uM. Each data point is representative
of the results for a set of chemically treated triplicates reported as
an average FOC i standard deviation determined at the 95%
confidence interval ................................................................ 74

Figure 3.4 Dose-response bioassay results for 4,4'-dichlorobiphenyl
(DMSO/BSA/PBS solvent) with 6 hours of incubation time for a
dose range of 0 to 260.87 uM. Each data point is representative
of the results for a set of chemically treated triplicates reported as
an average FOC 1 standard deviation determined at the 95%
confidence interval ................................................................ 75

xiv

Figure 3.5

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Dose-response bioassay results for 4,4'-dichlorobiphenyl
(DMSO/BSA/PBS solvent) with 24 hours of incubation time for a

dose range of 0 to 260.87 uM. Each data point is representative of

the results for a set of chemically treated triplicates reported as an

average FOC i standard deviation determined at the 95%

confidence interval ................................................................ 77

Structure of Methylene Blue ..................................................... 81

Methylene blue dye test results for Fenton’s reaction in Milli-Q
water. For test strips (A) control (no sample added) and (B) 40 uL

of Milli-Q water, no bleaching or discoloration of the methylene blue
dye is observed. Test strips with 40 uL of unquenched F enton’s
reaction mixture at (C) 15 minutes and (D) 30 minutes of reaction
indicate the presence of hydroxyl radicals by bleaching of the
methylene blue dye from dark blue to an almost white color with a
dark blue outline. Test strips with 40 pL of Fenton’s reaction mixture
quenched with (E) 30 drops and (F) 35 drops of 10% Nast3 indicate
the incomplete quenching and absence of hydroxyl radicals by very
slight bleaching and no bleaching, respectively .............................. 91

Thin layer chromatography results from “Benzoic Acid in an
Unquenched Fenton’s Reaction Mixture.” The left side of each TLC
plate was spotted with samples of standards. Figures 4.3A and 4.3B,
were spotted with the standards 9 mM benzoic acid (BA) in methanol
(MeOH) and 9 mM 4-hydroxybenzoic acid (HBA) in methanol,
respectively. The remainder of the TLC plates (Figures 4.3C-4.3F)
was spotted with 2 uL of a mixed standard consisting of 50 uL 9 mM
BA/MeOH and 25 uL 9 mM HBA/MeOH. The right side of each TLC
plate was spotted with a 3 uL sample of Fenton’s reaction mixture
from a particular reaction time. For Figures 4.3A to 4.3C, the

F enton’s sample was from 30 minutes of reaction, while for Figures
4.3D, 4.3B, and 4.3F, the sampling times were 60, 90, and

120 minutes, respectively ......................................................... 95

Wavelength scans (absorption spectra) of unquenched F enton’s
reaction mixture with a benzoic acid chemical probe. Wavelength
scans were performed over a wavelength range of 450 nm to 750 nm.
Each wavelength scan represents a Fenton’s reaction mixture sample
at a different reaction time ranging from 0 minutes to 120 minutes
after initiation of the F enton’s reaction. Absorbance vs. reaction
time of unquenched Fenton’s reaction mixture with a benzoic acid
chemical probe at the wavelength of 517 nm is shown in the inset.
Each data point is representative of the absorbance at 517 nm
obtained from the wavelength scan of the Fenton’s reaction mixture
at a particular reaction time. Polystyrene cuvettes with an optical

XV

Figure 4.5

Figure 4.6

Figure 4.7

Figure 5.1

Figure 5.2

pathlength of 10 mm were used ................................................. 99

Wavelength scans of the effect Of quenching an unquenched

Fenton’s reaction mixture containing a benzoic acid chemical probe

at 45 minutes of reaction time. Wavelength scans were performed

over a wavelength range of 400 nm to 750 nm. After performing a
wavelength scan on a 1000 uL sample of unquenched F enton’s

reaction mixture from 45 minutes of reaction time, 25 uL of 10%

sodium sulfite quencher was added to the sample. Following

mixing, a wavelength scan was performed on the quenched sample.
Polystyrene cuvettes with an optical pathlength of 10 mm were

used ................................................................................ 102

Wavelength scans (absorption spectra) of quenched Fenton’s reaction
mixture. Wavelength scans were performed over a wavelength range

of 400 nm to 750 nm. The “Pre-BA” wavelength scan represents a
sample of the Fenton’s reaction mixture prior to the benzoic acid

chemical probe addition, but after completion of quenching. The
remainder of the wavelength scans represents Fenton’s reaction

mixture samples at 0 to 90 minutes following the addition of benzoic

acid after the completion of quenching. Polystyrene cuvettes with an
optical pathlength of 10 mm were used ....................................... 107

Methylene blue dye test results evaluating (A) the affect of age of

the methylene blue dye test strip on test results by applying identical
samples of unquenched Fenton’s reaction solution to test strips aged

4 and 33 days, (B) the ability of 3% H202 to cause bleaching of the
methylene blue dye as compared to Milli-Q H20 and control (no

sample added) test strips, and (C) the affect of Milli-Q H2O pH on

the methylene blue dye test ..................................................... 108

Fenton’s remediation in Milli-Q water with a F e2+zH202 ratio of

1:20. Methylene blue dye test results for (A) control (no sample

added); (B) Milli-Q water; unquenched Fenton’s reaction mixture at

(C) 15 minutes and (D) 30 minutes of reaction; and Fenton’s

reaction mixture quenched with (E) 30 drops and (F) 35 drops of 10%
Na2SO3. All methylene blue dye tests were performed using 40 uL
samples ............................................................................ 123

F enton’s remediation in Milli-Q water with a Fe2+zH202 ratio of

1:40. Methylene blue dye test results for (A) control (no sample

added); (B) Milli-Q water; unquenched Fenton’s reaction mixture at

(C) 15 minutes, (D) 30 minutes, and (E) 60 minutes of reaction; and

F enton’s reaction mixture quenched with (F) 33 drops of 10%

Na2SO3. All methylene blue dye tests were performed using 40 pL
samples ............................................................................ 125

xvi

Figure 5.3 Dose-response GJIC bioassay results after a 30 minute incubation
time for a volume range of 0 to 300 uL of a solution resulting from
F enton’s reagent remediation with only Milli-Q water solvent
(no PCB), a Fe2+:H2O2 ratio of 1:5, and 1 hour reaction time. Each
data point is representative of the results for a set of chemically
treated triplicates reported as an average FOC i standard deviation
determined at the 95% confidence interval ................................... 129

Figure 5.4 Dose-response GJIC bioassay results after a 30 minute incubation
time for a volume range of 0 to 300 uL of a solution resulting from
Fenton’s reagent remediation with only Milli-Q water solvent
(no PCB), a Fe2+zH202 ratio of 1:20, and 1 hour reaction time. Each
data point is representative of the results for a set of chemically
treated triplicates reported as an average FOC i standard deviation
determined at the 95% confidence interval ................................... 130

Figure 5.5 Dose-response GJIC bioassay results after a 30 minute incubation
time for a volume range of 0 to 300 uL of a solution resulting from
Fenton’s reagent remediation with only Milli-Q water solvent
(no PCB), a Fe2+:H202 ratio of 1:40, and 1 hour reaction time. Each
data point is representative of the results for a set of chemically
treated triplicates reported as an average FOC 1: standard deviation
determined at the 95% confidence interval ................................... 131

Figure 6.1 Methylene blue dye test results for (A) control (no sample added),
(B) 40 uL of Milli-Q H20, (C) 40 uL of 100% acetonitrile (ACN),
(D) 40 p.L of 80/20 Milli-Q H2O/ACN, and (E) 40 uL of 50/50
Milli-Q H20/ACN ............................................................... 144

Figure 6.2 Methylene blue dye test results evaluating the effect of 80/20
Milli-Q H2O/ACN solvent at pH values (A) 3.6, (B) 6.8, (C) 8.3,
and (D) 10.0. All methylene blue dye tests were performed using
40 uL samples .................................................................... 146

Figure 6.3 Fenton’s remediation of 80/20 Milli-Q H2O/ACN solvent with a
Fe2+:H2O2 molar ratio of 1:20. Methylene blue dye test results for
(A) control (no sample added); (B) Milli-Q water; (C) 80/20
Milli-Q H20/ACN; unquenched Fenton’s reaction mixture at
(D) 15 minutes, (E) 30 minutes, and (F) 60 minutes; and Fenton’s
reaction mixture quenched with (G) 6 drops and (H) 11 drops of
10% Na2803. All methylene blue dye tests were performed using
40 uL samples ................. . ................................................... 148

xvii

Figure 6.4

Figure 6.5

Figure 6.6

Figure 7.1

Figure 7.2

F enton’s remediation of 50/50 Milli-Q H2O/ACN solvent with a

F e2+zH2O2 molar ratio of 1:20. Methylene blue dye test results for

(A) control (no sample added); (B) Milli-Q water; (C) 50/50

Milli-Q H20/ACN; unquenched Fenton’s reaction mixture at

(D) 15 minutes, (E) 30 minutes, and (F) 60 minutes; and Fenton’s

reaction mixture quenched with (G) 6 drops and (H) 8 drops of

10% Na2803. All methylene blue dye tests were performed using

40 uL samples .................................................................... 153

Dose-response GJIC bioassay results with a 30 minute incubation

time for a volume range of 0 to 150 uL of a solution resulting fi'om
Fenton’s reagent remediation with only 80/20 Milli-Q water/ACN

solvent (no PCB), a Fe2+zH2O2 ratio of 1:20, and 60 minutes reaction

time. Each data point is representative of the results for a set of
chemically treated triplicates reported as an average FOC i standard
deviation determined at the 95% confidence interval ....................... 157

Dose-response GJIC bioassay results with a 30 minute incubation

time for a volume range of 0 to 60 uL of a solution resulting from

F enton’s reagent remediation with only 50/50 Milli-Q water/ACN

solvent (no PCB), a Fe2+zH2O2 ratio of 1:20, and 60 minutes reaction

time. Each data point is representative of the results for a set of
chemically treated triplicates reported as an average FOC i standard
deviation determined at the 95% confidence interval ....................... 158

F enton’s remediation of a solution of 4,4'—dichlorobiphenyl in 50/50
Milli-Q H20/ACN. Methylene blue dye test results for (A) control
(no sample added); (B) Milli-Q water; (C) 50/50 Milli-Q H2O/ACN
solvent; unquenched Fenton’s reaction mixture at (D) 15 minutes,
(E) 30 minutes, and (F) 60 minutes; and Fenton’s reaction mixture
quenched with (G) 6 drops and (H) 8 drops of 10% Na2SO3. All
methylene blue dye tests were performed using 40 uL samples. The
F enton’s reaction conditions were pH 3.0, temperature 23.0 °C,
Fe2+zH2O2 molar ratio 1:20, initial Fe2+ concentration 0.15 mM, and
initial H202 concentration 3 mM. After a 60 minute remediation
reaction period, the Fenton’s reaction was quenched with a 10%
Na2S03 solution (w/v). Following quenching with 10% Na2SO3,
the F enton’s reaction mixture pH increased to 9.0 and the
temperature remained at 23.0 °C .............................................. 176

Dose-response GJ 1C bioassay results with a 30 minute incubation
time for a volume range of 0 to 60 uL of a solution resulting from
Fenton’s remediation of 4,4'-dichlorobiphenyl in 50/50 Milli-Q
H2O/ACN solvent. Each data point is representative of the results
for a set of chemically treated triplicates reported as an average

FOC :1: standard deviation determined at the 95% confidence interval.

xviii

Figure 7.3

Figure 8.1

Figure 8.2

Figure 8.3

Figure 8.4

Figure 8.5

The F enton’s reaction conditions were pH 3.0, temperature 23.0 °C,
FeZ+zH2O2 molar ratio 1:20, initial F e2+ concentration 0.15 mM, and
initial H202 concentration 3 mM. After a 60 minute remediation

reaction period, the Fenton’s reaction was quenched with a 10%

Na2S03 solution (w/v). Following quenching with 10% Na2SO3, the
Fenton’s reaction mixture pH increased to 9.0 and the temperature
remained at 23.0 °C. The reaction mixture was then filtered through

a 1.0 pm glass fiber filter and the filtrate was adjusted to pH 7 ........... 181

Time-response GJIC bioassay results for a 60 uL test volume of a
solution resulting from Fenton’s remediation of 4,4'-dichlorobiphenyl
in 50/50 Milli-Q H2O/ACN solvent. Time of chemical exposure
varied from 0 minutes to 1440 minutes (24 hours). Each data point is
representative of the results for a set of chemically treated triplicates
reported as an average FOC :I: standard deviation determined at the
95% confidence interval. Data points after the break correspond to
240 minutes, 480 minutes, 720 minutes, and 1440 minutes of chemical
exposure time, respectively. The Fenton’s reaction conditions were
pH 3.0, temperature 23.0 °C, Fe2+zH202 molar ratio 1:20, initial Fc2+
concentration 0.15 mM, and initial H202 concentration 3 mM. After

a 60 minute remediation reaction period, the F enton’s reaction was
quenched with a 10% Na2SO3 solution (w/v). Following quenching
with 10% Na2S03, the Fenton’s reaction mixture pH increased to 9.0
and the temperature remained at 23.0 °C. The reaction mixture was
then filtered through a 1.0 pm glass fiber filter and the filtrate was
adjusted to pH 7 .................................................................. 184

Gas chromatogram for the blank (isooctane) ................................. 206
Gas chromatogram for 220 ppm 3~chloro-2-biphenylol/isooctarle ........ 208

Gas chromatogram for 200 ppm
4,4'-dichloro-3—biphenylol/isooctane .......................................... 210

Comparison of the isooctane extraction GC/ECD results for

efficiency of recovery of 4,4'—dichlorobiphenyl and the calibration

curve for 4,4'DCBP/isooctane. The isooctane extraction GC/ECD

results were graphed based on the expected extraction

concentrations .................................................................... 213

Comparison of the relationships between the percent efficiency of
recovery of 4,4'DCBP by isooctane extraction, the calculated

expected extraction concentrations based on the original volumes

of 4,4'DCBP/ACN stock solution prepared for extraction, and the

percent of ACN in the dilution water of the extraction process ........... 216

xix

Figure 8.6

Figure 8.7

Figure 8.8

Figure 8.9

Figure 8.10

Figure 8.11

Figure 8.12

Figure 8.13

Figure 8.14

Fenton’s remediation of a solution of 4,4'-dichlorobiphenyl in 50/50
Milli-Q H2O/ACN. Methylene blue dye test results for (A) control

(no sample added), (B) 40 uL of Milli-Q water, (C) 40 uL of 50/50
Milli-Q H2O/ACN solvent, and (D) 40 uL of the reaction mixture

from “0 minutes” of remediation .............................................. 219

F enton’s remediation of a solution of 4,4’-dichlorobiphenyl in 50/ 50
Milli-Q H2O/ACN. Methylene blue dye test results for (A) control
(no sample added); (B) Milli-Q water; (C) 50/50 Milli-Q H2O/ACN
solvent; unquenched Fenton’s reaction mixture at (D) 15 minutes,
(E) 30 minutes, and (F) 60 minutes; and F enton’s reaction mixture
quenched with (G) 6 drops and (H) 8 drops of 10% Na2S03. All

methylene blue dye tests were performed using 40 uL samples .......... 222
Representative calibration curve gas chromatogram for 40 ppm
4,4’DCBP/isooctane ............................................................. 227
Representative calibration curve gas chromatogram for 80 ppm
4,4’DCBP/isooctane ............................................................. 229
Representative calibration curve gas chromatogram for 160 ppm
4,4’DCBP/isooctane ............................................................. 23 1
Calibration curve for 4,4’DCBP/isooctane ................................... 234

Gas chromatogram for the “78.05 ppm” 4,4'DCBP/isooctane

extraction sample from the F enton’s remediation solution reacted for

60 minutes (pH 3.0, temperature 23.0 °C, Fe2+zH2O2 molar ratio 1:20,
initial F e2+ concentration 0.15 mM, and initial H202 concentration

3 mM) .............................................................................. 236

Gas chromatogram for the “156.1 ppm” 4,4'DCBP/isooctane

extraction sample from the F enton’s remediation solution reacted

for 60 minutes (pH 3.0, temperature 23.0 °C, Fe2+zH2O2 molar ratio

1:20, initial Fe2+ concentration 0.15 mM, and initial H202

concentration 3 mM) ............................................................ 238

Graph of the average percent of 4,4 '-dichlorobiphenyl remaining
following F enton’s remediation vs. the remediation time .................. 243

ACN

AN OVA
BA

2BP
2,2'BP
3BP
3,3'BP
BSA
3C2BP

CI
4,4’DCBP
4,4'DC3BP
DMSO
ECD

F BS

FOC
GC/ECD
GJIC
HBA

4-HBA

ABBREVIATIONS

Acetonitrile

Analysis of Variance

Benzoic Acid

2—Biphenylol

2,2’-Biphenyldiol

3-Biphenylol

3,3'-Biphenyldiol

Bovine Serum Albumin
3—Chloro-2-biphenylol

Confidence Interval

4,4’-Dichlorobiphenyl
4,4'-Dichloro-3-biphenylol

Dimethyl Sulfoxide

Electron Capture Detector

Fetal Bovine Serum

Fraction of the Control

Gas Chromatography/Electron Capture Detection
Gap Junctional Intercellular Communication
Hydroxylated Benzoic Acid
4-Hydroxybenzoic Acid

Methylene Blue

xxi

MeOH

OH-BP

OH—PCB

PBS

PCB

SD

SL/DT

TLC

Methanol

Hydroxylated Biphenyl

Hydroxylated Polychlorinated Biphenyl
Phosphate Buffered Saline
Polychlorinated Biphenyl

Standard Deviation
Scrape-Loading/Dye Transfer

Thin—Layer Chromatography

xxii

Chapter 1

Introduction

1.1 Background and Environmental Significance

Polychlorinated biphenyls (PCBs) are synthetic organic compounds with a
biphenyl backbone consisting of two hexagonal “rings” of carbon atoms connected by
carbon-carbon bonds. Polychlorinated biphenyls have between 1 and 10 chlorine atoms
substituted for hydrogen atoms on the biphenyl rings and, therefore, are part of a larger
group of chemicals known as halogenated aromatic compounds. The combinations of the
chlorine atoms on the biphenyl molecule result in 209 possible chemical structures
known as congeners. Chemical properties, such as nonflammability, chemical and
thermal stability, low reactivity, miscibility with organic compounds, and excellent
dielectric properties, made PCBs ideal for use in numerous commercial and industrial
products, such as transformers, capacitors, light ballasts, paints, adhesives, dyes, plastics,
hydraulic fluids, flame retardants, and lubricants (1, 2). PCBs are moderately volatile
from water and soil, have melting points of 340 °C to 375 °C, and are non-polar.

PCBs were manufactured in the United States from 1929 to 1977. The decision to
ban commercial production, processing, and distribution of all PCBs in the United States
was reached in 1976, following a series of events which alerted worldwide concern about
the potential health effects of PCBs. Manufacture of PCBs in the United States ceased in
1977 under the Toxic Substances Control Act. PCBs were banned by many countries in
the late 1970’s and a global ban occurred in 2001 under the Stockholm Convention on

persistent organic pollutants.

PCB-contaminated soils are classified into 3 groups based on the levels of
contamination and in the order of decreasing severity of disposal restrictions: (i) greater
than 500 ppm as PCB material, (ii) 50 to 500 ppm as PCB-contaminated material, and
(iii) less than 50 ppm as waste material (3). Due to the severity of disposal restrictions
applied to materials with high levels of PCB contamination, an incentive exists for PCB
waste generators to develop methods to reduce the PCB level to the range where the
wastes can be disposed of under less severe restrictions. Drinking water standards for
chemicals that are implicated as causing health problems were developed as a result of
the Safe Drinking Water Act. Maximum Contaminant Level Goals (MCLG) are non-
enforceable levels based solely on possible health risks and exposure. The MCLG for
PCBs has been set at zero because the EPA believes this level of protection would not
cause any potential health problems. The EPA has set an enforceable standard, based on
the MCLG, called the Maximum Contaminant Level (MCL), which is set as close to the
MCLGs as possible, considering the ability of public water systems to detect and remove
contaminants using suitable treatment technologies. The MCL for PCBs is set at 0.5
parts per billion (ppb) (4).

Although production of PCBs has been discontinued and disposal of PCBs fiom
existing sources has been highly regulated, PCBs continue to be a concern due to their
persistence in the environment, slow rate of biodegradation, ability to bioaccumulate, and
the potential impact on humans and the environment. PCBs have been found to exist in
almost every component of the global ecosystem including air, water, sediments, and
soils (1). Environmental cycling of PCBs previously introduced into the environment can

occur by volatilization from ground surfaces (water, soil) into the atmosphere, removal

from the atmosphere via wet/dry deposition, and revolatilization (4). Potential
environmental sources of PCBs include past open uncontrolled uses, past disposal
practices, illegal disposal, and accidental releases (5, 6). Humans and wildlife can be
exposed to PCBs either directly from contact with contaminated air, sediments, or water
or indirectly through food. The non-polar, lipophilic physical properties of PCBs, as well
as their resistance to biochemical degradation, tend to cause their accumulation in fatty
tissues of humans and wildlife (2). Numerous human health effects, including
reproductive disorders, embryotoxicity, oncogenicity, estrogenic endocrine disruption,
wasting syndrome, reduced body weight, immunotoxicity, vitamin A deficiency, thyroid
deficiency, and reduced birth weight, have been attributed to exposure to PCBs (5, 7, 2).
The EPA has classified all PCBs as Group BZ, probable human carcinogens (8).

A number of PCBs are potent inducers of hepatic and extrahepatic xenobiotic
metabolizing enzymes and the activity of the individual compounds are remarkably
dependent on structure. They can be divided schematically into three groups. Group 1
consists of Phenobarbital (PB)-type inducers, Group 2 consists of 3-methylcholanthrene
(3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-type inducers, and Group 3
consists of inducers of both Groups 1 and 2 and is called mixed—type inducers. The most
toxic congeners, 3,4,3'4'-tetrachlorobiphenyl, 3,4,5,3'4'-pentachlorobiphenyl, and
3,4,5,3'4'5'-hexachlorobiphenyl are lacking chlorosubstitution in the ortho positions.
These congeners might form coplanar conformation and are thereby approximate
isostereomers of 2,3,7,8-trichlorodibenzodioxin (TCDD). They also elicit biological
effects comparable to those reported for TCDD, including enzyme induction pattern of

the MC-type, both in Vitro and in vivo. Therefore, the main structural characteristics are

chloro substituents at both para positions, chloro substitution in at least one meta position
of both phenyl rings and no ortho substituents. TCDD has been shown to not inhibit gap
junction intercellular communication (measured as metabolic cooperation between
Chinese Hamster V-79 cells originally derived from lung tissue). TCDD-like PCBs and
other PCBs lacking ortho substituted chlorine atoms, have a coplanar conformation and
also do not inhibit gap junction intercellular communication (9).

Introduction of one or more chlorosubstituents in the ortho position results in a
decreased degree of coplanarity between the two phenyl rings due to steric interactions.
Mono- and diorthO-chloro substitutions of the three most toxic congeners give toxic
effects that resemble the toxicity of TCDD, qualitatively. However, the liver enzyme
induction pattern is often of the mixed type. Gap junction intercellular communication
was totally inhibited by all PCBs containing at least one chloro substituent in the ortho
position with the exception of one highly chlorinated congener, 2,3,4,5,3',4',5'-
heptachlorobiphenyl. One hypothesis for why 2,3,4,5,3',4',5'-heptachlorobiphenyl did
not totally inhibit gap junction intercellular communication is that highly chlorinated
congeners require more ortho substituents to inhibit gap junction intercellular
communication. The potential for inhibition of gap junction intercellular communication
generally increased with increasing numbers of ortho-substituted chlorine atoms (9).
Results show that substitution in the ortho position from the carbon bridge is essential
and at least one chloro substituent in the ortho position is necessary for the ability to
inhibit gap junction intercellular communication (9). However, the total number of
substitutions may not be crucial for the ability to inhibit gap junction intercellular

communication (9). The steric interactions between more than two ortho substituents

would markedly inhibit biphenyl ring coplanarity (thus, making these compounds
noncoplanar). Many compounds of this group have been found to be PB-type inducers
(9).

Brown et al. (10) studied the ability of polychlorinated biphenyls (PCBs) to
stimulate polymorphonuclear leukocytes (neutrophils) in vitro in Sprague-Dawley rats.
Congeners which are noncoplanar stimulated neutrophil 02' (superoxide) production,
whereas coplanar congeners with high affinity for the arylhydrocarbon receptor (AhR) do

not and might even inhibit this response.

1.2 Remediation of Polychlorinated Biphenyls Using Fenton’s Reagent

A variety of physical, chemical, and biological processes have been investigated
for the remediation of PCBs. Chemical oxidation could be used as a pretreatment step to
enhance the biodegradability of PCBs or as a primary method of remediation. One form
Of chemical oxidation, Fenton’s reagent, has been found to be an effective method of
remediating PCB contaminated soils and aqueous solutions through oxidation by
hydroxyl radicals (3, 11). In the F enton’s reagent process, highly reactive hydroxyl
radicals are produced by the oxidation of ferrous iron and the reduction of hydrogen
peroxide (3). The hydroxyl radical is one of the most reactive chemical species known
and is second only to elemental fluorine in its relative oxidation power. The classical
procedure of the F enton’s reagent treatment consists of the addition of hydrogen peroxide
to a solution or suspension of compounds (reactive chemicals) in the presence of ferrous
iron (12) at pH 2-3. Numerous reactions might be involved with F enton’s reagent,

depending on the nature of the reacting substrates. One potential pathway, presented by

Trapido et al. (12), involving the abstraction of a hydrogen atom and initiating a radical

chain oxidation is shown below:

Fe2+ + H202 —+ Fe3+ + H0‘ + -OH (1)
RH + -OH —> R- + H20 (2)
R- + H202 —> ROH + -OH (3)
R0 + 02 —> R00- (4)

The oxidation efficiency of the Fenton’s type reactions depends on the Fe2+zH2O2 ratio

and the pH value. The optimal pH for the Fenton’s reagent reaction efficiency has been

shown to be between pH 3 and 5. At more basic pH values, the iron is converted from a

hydrated ferrous form to a colloidal ferric form, thereby causing a decrease in the

effectiveness of the reaction and the formation of ferric hydroxide (13-15).

Advantages of Fenton’s reagent remediation are as follows:

a) The hydroxyl radical is a powerful oxidant. It is twice as reactive as chlorine and
second only to fluorine among common oxidants in the oxidation potential series.

b) Fenton’s remediation can detoxify a broad range of organic wastes and is effective
over a wide range of contaminant concentrations (1 3).

0) Hydrogen peroxide, unlike chlorine, does not require large cylinders for storage and
does not pose the dangers of chlorine gas. It can be stored on-site in quantities
appropriate to user requirements.

(1) Both iron and hydrogen peroxide are cheap and environmentally friendly. Unreacted
hydrogen peroxide degrades in the environment to oxygen and water.

e) Fenton’s remediation is effective as a pretreatment step that increases the rate and

efficiency of biological degradation of contaminants. Partial chemical oxidation

enhances water solubility of the organic compounds and increases biosusceptibility,
facilitates microbial action, and increases the biodegradation rate. (16)

Disadvantages of F enton’s reagent remediation are as follows:

a) Since the reaction requires acidic conditions, the method of Fenton’s oxidation might
not be applicable to alkaline solutions or sludges with high buffering capacities (1 7).

b) Production of iron sludge, which must be disposed of.

c) Hydroxyl radicals are non-specific in nature and might react with non-pollutant
species present at a higher concentration. (13)

d) Effectiveness of the reaction is influenced by pH, iron complexation, iron solubility,
the F e2+zH2O2 ratio, and iron redox cycling between the +2 and +3 states.

e) Potential production of remediation byproducts with equal or greater toxicity than the
original contaminant.

f) In some situations a rise in temperature might occur as the reaction proceeds.

g) Hydrogen peroxide is highly reactive. The reaction can be vigorous, with the rapid
evolution of oxygen, steam, and carbon dioxide. Allowances should be made to
ensure adequate venting of these gases.

When applied to PCBs, this process would result in the addition of a hydroxyl
group to nonhalogenated sites and the production of hydroxylated polychlorinated
biphenyls (11). Sedlak et al. (11) reported that dechlorination reactions are insignificant
relative to hydroxylation reactions in the initial attack of hydroxyl radicals on PCBs;
however, further reactions might occur, resulting in dechlorination and production of
hydroxylated biphenyls. Basu et al. (18) showed that reactions of F enton’s reagent with

aqueous phase 2,4,6-trichlorophenol are also associated with the immediate release of

chlorine atoms into free chloride ions in solution. In a study on F enton’s oxidation of 2-
chlorobiphenyl in aqueous solution, Sedlak et al. (11) suggested 5-hydroxy-2-
chlorobiphenyl (or 2-chloro-5-biphenylol) as one of the byproducts.

Current remediation practices often emphasize the disappearance of the parent
compound to at or below the regulatory limits, but often disregard the importance of
reducing the overall toxicity. Since remediation byproducts can exhibit equal or greater
toxicity than the parent compound, it is important to consider in a remediation process
not only the removal of the parent compound, but also the toxicological impact of the
remediation byproducts (I 9). Although the reaction of F enton’s reagent with various
PCBs has been investigated (11, 3), the subsequent toxicity and characterization of

remediation byproducts have not been extensively studied.

1.3 Toxicity of Hydroxylated Polychlorinated Biphenyls (OH-PCBs)

Within organisms, PCBs are metabolized by a diverse enzyme system, the
cytochrome P450 monooxygenases, into hydroxylated polychlorinated biphenyls (OH-
PCBs) (20). The metabolite formed is dependent upon the chlorine arrangement in the
PCB and the type of enzyme involved (21). Less halogenated PCBs are readily
metabolized to monohydroxy derivatives, which might be further metabolized to ortho-
or para-dihydroxy metabolites and quinones (22). OH-PCBs have been detected in
human organs, blood, fatty tissue, and milk, as well as in fish and wildlife (23). OH-
PCBs have been shown to be transferred by placental transfer to the fetus both in humans
and in animals (24). Several high molecular weight hydroxylated PCB metabolites have

been found to be strongly and selectively accumulated in mammalian tissues, including

human blood (22). Although lower molecular weight OH-PCBs have been considered to
be nonpersistent, they may occur transiently during episodic exposures to PCBs (22).
Some OH-PCBs might have a greater potential to disrupt biological systems than their
parent compound. (21 ). OH-PCBs have been shown to have estrogenic and anti-
estrogenic effects, can disrupt thyroxin and vitamin A transport, and might lead to
adverse neurodevelopmental effects (20, 25). Since OH—PCBs are relatively stable in
mammalian systems, they are eliminated mainly by excretion in urine and feces and are
detected as residues in the environment (25). Based upon 1996 amendments to the Safe
Drinking Water and Food Quality Protection Act, OH-PCBs are among the pollutants
requiring monitoring as estrogenic substances in drinking water (25). The OH-PCBs
produced from PCB remediation processes, such as the F enton’s reagent procedure, are a
potential environmental concern, thereby justifying toxicological evaluation.

Few studies have been performed regarding the effects of OH-PCBs on GJIC and
none have investigated the OH-PCBs examined in this dissertation. Machala et al. (22)
observed inhibition of GJIC for a selected group of OH-PCBs, with the strongest
inhibition for OH-PCBs with the highest molecular weight. The majority of the mono-
and dihydroxy-PCBs studied inhibited GJIC in vitro in micromolar concentrations. No
cytotoxic efiects of the tested compounds were observed with 30 minutes and 24 hours of

exposure at concentrations up to 100 uM.

1.4 Gap Junctional Intercellular Communication (GJIC) as a Marker of Toxicity
When considering the toxicity of a chemical, the potential of that chemical to

cause cancer is a major concern. Three means exist by which toxic chemicals can alter

the functioning of cells. “Genotoxicity” or mutagenesis is when a toxic chemical alters
the genetic information of a cell (the DNA code) or the number or structure of the
chromosomes. Cytotoxicity is when a toxic chemical kills a cell by a necrotic
mechanism (cell basically dies because of membrane damage, critical enzymes or
proteins are destroyed) or an “apoptotic” mechanism (a chemical causes the cell to
commit suicide (programmed cell death)). Epigenetic toxicity is when a nonmutagenic
and noncytotoxic chemical alters the expression of normal genes causing inappropriate
“turning on or off” of genes in a cell at the wrong place at the wrong time (26). The
alteration of the expression of genes can occur at the transcriptional, translational, or
posttranslational levels (19). The ability to disrupt gap junctional intercellular
communication (GJIC) is indicative of one form of a chemical’s epigenetic toxicity.
Epigenetic toxicity can be evaluated using the GJIC assay, a nongenotoxic assay that
measures the level of cell—cell communication (or GJIC).

Gap junctions are organized collections of protein channels in the cell membrane
that allow ions and small molecules (S 1200 Da) to traverse passively between the cells
that they connect (2 7). GJIC, which is required for cellular growth control,
developmental and differentiation processes, synchronization, and metabolic regulation,
results fi'om this passive transfer of ions and small molecules through the gap junctions
(28). Gap junctions exist in all metazoans (multi-cellular organisms) and in almost all
cell types in these organisms. Some cells that lack gap junctions include skeletal muscle,
red blood cells, and free standing cells such as circulating lymphocytes (27). The
majority of cancer cells have dysfunctional gap junctional intercellular communication

(29). Also, many non-mutagenic but toxic chemicals have been shown to inhibit GJIC

10

and to promote the growth of premalignant tumor cells (29). It has been suggested by
Trosko et al. (30) that the reversible inhibition of GJIC by endogenous or exogenous
tumor promoters is responsible for the tumor promotion phase of carcinogenesis.

Some PCBs have been shown to be epigenetically toxic by being capable of disrupting
GJIC (I, 9, 31, 32). PCBs with coplanar conformations appear to be less likely to inhibit
GJIC, whereas decreased coplanarity of the PCB increases the chances of inhibition of
GJIC (9, 31, 32). No literature could be found regarding the effects of hydroxylated
biphenyls (OH-BPS) on GJIC. By performing bioassays that measure the effect of

chemical exposure on GJIC, an evaluation of the potential cancer risk is possible.

1.5 Hypotheses and Objectives

The dissertation research was performed with the hypothesis that F enton’s
remediation of the parent PCB, 4,4'-dichlorobiphenyl (4,4'DCBP), under a set of selected
conditions would result in a final remediation solution that indicates a decrease in the
concentration of the parent PCB, is more toxic than the parent PCB, and exhibits some of
the toxicological properties demonstrated by the selected potential remediation byproduct
standards (particularly 4,4'-dichloro-3 -biphenylol).

The dissertation research was performed with the following Obj ectives:
a) To determine whether there is any correlation between the chemical and structural

properties of OH-PCBs and OH-BPs, as potential byproducts of remediation of PCBs

with Fenton’s reagent, and their observed epigenetic toxicity and cytotoxicity.

11

b)

d)

g)

h)

To evaluate the toxicity of the parent PCB, 4,4'—dichlorobiphenyl in rat liver epithelial
cells using a nongenotoxic bioassay that determines the in vitro modulation of GJIC
as a measure of the epigenetic toxicity.

To develop a new test that qualitatively indicates the presence of hydroxyl radicals in
aqueous samples and evaluate its applicability for the detection of hydroxyl radicals
in a F enton’s reaction aqueous solution.

To verify that the reaction of Fenton’s reagent with the solvent for remediation of the
parent PCB does not result in any toxic byproducts, and select a suitable solvent for
the remediation of the parent PCB.

To develop a procedure for F enton’s remediation of the parent PCB.

TO perform F enton’s remediation of 4,4'DCBP in the selected solvent, followed by
examination of the toxicity of the final F enton’s remediation solution and
disappearance of 4,4’DCBP.

To compare the toxicity of the final F enton’s remediation solution to the toxicity
observed for the potential remediation byproducts and parent PCB.

To analyze the gas chromatograms from the final Fenton’s remediation solution for
the presence of any remediation byproduct peaks.

To determine the retention times for the chlorinated potential remediation byproducts
previously investigated in the toxicology studies and compare the retention patterns to

those of the extracted samples.

12

1.6 Organization of Dissertation

The research performed for the dissertation was separated into four main divisions.
The first division, comprised of Chapters 2 and 3, involved toxicology studies of six
selected potential Fenton’s remediation byproducts and the selected parent PCB (4,4'-
dichlorobiphenyl). The objective of the research described in Chapter 2 was to determine
whether there is any correlation between the chemical properties (including structure) of
OH-PCBs and OH-BPs and their observed epigenetic toxicity and cytotoxicity. Six
commercially available chemicals considered to be representative of OH-BPs and OH-
PCBs, which are potential byproducts of remediation of PCBs with Fenton’s reagent,
were selected for study. These six chemicals were 2-biphenylol (2BP), 3-biphenylol
(3BP), 2,2'-biphenyldiol (2,2’BP), 3,3'-biphenyldiol (3,3'BP), 3-chloro-2-biphenylol
(3C2BP), and 4,4'-dichloro—3-biphenylol (4,4’DC3BP). The toxicity of these chemicals
was evaluated in rat liver epithelial cells using a nongenotoxic bioassay that determines
the in vitro modulation of GJIC as a measure of the epigenetic toxicity. For each
chemical the dose-response, time-response, time of recovery, and cytotoxicity were
investigated. In view of the cytotoxicity and GJIC inhibitory effects observed for 4,4'-
dichloro-3-biphenylol (4,4'DC3BP), the PCB congener 4,4'-dichlorobiphenyl
(4,4'DCBP) was selected for F enton’s reagent remediation studies. In Chapter 3, the
toxicity of the parent PCB (4,4'DCBP) for the Fenton’s remediation studies was
evaluated in rat liver epithelial cells using a nongenotoxic bioassay that determines the in
vitro modulation of GJIC as a measure of the epigenetic toxicity.

The methylene blue (MB) dye test was developed as a new test that qualitatively

indicates the presence of hydroxyl radicals through an immediate, distinct bleaching of

13

the MB dye on a paper test strip after applying an aqueous sample containing hydroxyl
radicals. The second division, comprised of Chapter 4, tested the applicability of the MB
dye test for the detection of hydroxyl radicals in a F enton’s reaction aqueous solution and
verified the results by benzoic acid chemical probe hydroxyl radical detection methods
using thin layer chromatography and spectrophotometric wavelength scans. For Fenton’s
remediation experiments throughout the dissertation research, MB dye tests were
performed during the Fenton’s reaction to verify the formation of hydroxyl radicals and
during the quenching process to verify completion of quenching (absence of hydroxyl
radicals).

The third division, comprised of Chapters 5 and 6, verified that the reaction of
F enton’s reagent with the solvents (Milli-Q H2O alone, 80/20 Milli-Q H2O/ACN (by
volume), and 50/50 Milli-Q H2O/ACN (by volume)) in the absence of 4,4’DCBP, does
not result in any toxic byproducts. If no toxic response occurs as a result of a reaction of
F enton’s reagent with the solvent, any toxicity resulting from the Fenton’s reagent
remediation of 4,4’DCBP can be assumed to be independent of the solvent. Additionally,
this series of experiments allowed for the development of a remediation procedure that
later would be applied to 4,4 'DCBP. Evaluation of whether Fenton’s reagent remediation
in water alone resulted in any toxicity is discussed in Chapter 5. Although 4,4'DCBP is
insoluble in water alone, since water constituted some part of the solvent used for
dissolving 4,4'DCBP in remediation, it was important to investigate whether F enton’s
reagent remediation with water alone resulted in any toxicity. Three molar Fe2+zH2O2
ratios, 1:5, 1:20, and 1:40, were investigated in this section of research to determine the

ratio that would be used for F enton’s remediation of 4,4'DCBP. In Chapter 6, Fenton’s

14

remediation of the solvents 80/20 Milli-Q H2O/ACN and 50/50 Milli-Q H2O/ACN (by
volume) were investigated to determine the toxicological effect of ACN in combination
with water as a solvent in remediation. Based on the results of F enton’s remediation in
Milli-Q water, discussed in detail in Chapter 5, an F e2+zH2O2 ratio of 1:20 was selected
for each Fenton’s remediation experiment in Chapter 6. In both Chapters 5 and 6, the
toxicity of the resulting final F enton’s remediation solutions was evaluated in rat liver
epithelial cells, using a nongenotoxic bioassay that determines the in vitro modulation of
gap junctional intercellular communication (GJIC) as a measure of the epigenetic toxicity.
The fourth division, comprised of Chapters 7 and 8, involved F enton’s

remediation of 4,4'DCBP in 50/50 Milli-Q H2O/ACN, followed by examination of the

toxicity of the final Fenton’s remediation solution and disappearance of 4,4'DCBP as a
result of Fenton’s remediation. The Fenton’s reaction conditions were pH 3.0,
temperature 23.0 °C, Fe2+zH2O2 molar ratio 1:20, initial Fe2+ concentration 0.15 mM, and
initial H202 concentration 3 mM. The pre-remediation concentration of 4,4’DCBP in the
reaction mixture was 2 mM (or 446 ppm). After a 60 minute remediation reaction period,
the F enton’s reaction was quenched with a 10% Na2S03 solution (w/v). Following
quenching with 10% Na2SO3, the Fenton’s reaction mixture pH was adjusted to 9.0
(unless the pH was already greater than or equal to pH 9.0) and the temperature remained
at 23.0 °C. The reaction mixture was then filtered through a 1.0 pm glass fiber filter and
the filtrate was adjusted to pH 7.0. In Chapter 7, the toxicity of the resulting final
Fenton’s remediation solution was evaluated in rat liver epithelial cells, using a
nongenotoxic bioassay that determines the in vitro modulation of gap junctional

intercellular communication (GJIC) as a measure of the epi genetic toxicity. The toxicity

15

of the final Fenton’s remediation solution was compared to the toxicity observed for the
potential remediation byproducts and 4,4’DCBP studied in Chapters 2 and 3, respectively.
Chapter 8 determined the disappearance of 4,4'DCBP throughout the Fenton’s
remediation process for the reaction times of 0, 15 , 30, and 60 minutes. Samples of the
Fenton’s remediation mixture extracted with an isooctane liquid-liquid extraction method
were analyzed by gas chromatography/electron capture detection (GC/ECD) to quantitate
the disappearance of the parent PCB, 4,4'DCBP, over the period of remediation. The gas
chromatograms were analyzed for the presence of any remediation byproduct peaks. In
addition, retention times were determined for the chlorinated potential remediation
byproducts previously investigated in toxicology studies (Chapter 2) and the retention

patterns were compared to those of the extracted samples.

16

1.7 References

1.

10.

11.

Safe, S. Polychlorinated biphenyls (PCBs): Mutagenicity and Carcinogenicity. Mutat.
Res. 1989, 220, 31-47.

National Research Council. A Risk—Management Strategy for PCB-Contaminated
Sediments. National Academy Press: Washington, DC, 2001.

Sato, 0; Leung, S.W.; Bell, H.; Burkett, W.A.; Watts, RJ. Decomposition of
Perchloroethylene and Polychlorinated Biphenyls with F enton's Reagent (Chapter
16). Emerging Technologies in Hazardous Waste Management 111 (ACS Symposium
Series), American Chemical Society: Atlanta, GA, 1991.

US. Environmental Protection Agency. Technical Factsheet on:
POLYCHLROINATED BIPHENYLS (PCBs). http//www.epa.gov/OGWDW/dwh/t-
soc/pcbs.html (accessed March 2008).

Danse, I.R.; Jaeger, R.J.; Kava, R.; Kroger, M.; London, W.M.; Lu, F.C.; Maickel,
R.P.; McKetta, J .J .; Newell, G.W.; Shindell, S.; Stare, F.J.; Whelan, E. M. Review:
Position Paper of the American Council on Science and Health: Public Health
Concerns about Environmental Polychlorinated Biphenyls (PCBs). Ecotoxicol.
Environ. Saf 1997, 38, 71-84.

Erickson, M. D. Analytical Chemistry of PCBs. CRC Lewis Publishers: Argonne, IL,
1997.

Soto, A.M.; Sonnenschein, C.; Chung, K.L.; Fernandez, M.F.; Olea, N.; Serrano, F0.
The E—SCREEN Assay as a Tool to Identify Estrogens: An Update on Estrogenic
Environmental Pollutants Environ. Health Perspect. 1995, 103 (7), 113-122.

US. Environmental Protection Agency. Integrated Risk Information system (IRIS)
on PCBs. Environmental Criteria and Assessment Office, Office of Health and
Environmental Assessment, Office of Research and Development: Cincinnati, OH,
1994.

Hemming, H.; Wamgard, L.; Ahlborg, U.G. Inhibition of Dye Transfer in Rat Liver
WB Cell Culture by Polychlorinated Biphenyls. Pharmacol. Toxicol. 1991, 69 (6),
416-420.

Brown, A.P.; Olivero-Verbel, J .; Holden, W.L.; Ganey, P.E. Neutrophil activation by
polychlorinated biphenyls: structure-activity relationship. T oxicol. Sci. 1998, 46 (2),
308-3 16.

Sedlak, D.L.; Andren, A.W. Aqueous-Phase Oxidation of Polychlorinated Biphenyls
by Hydroxyl Radicals. Environ. Sci. T echnol. 1991, 25 (8), 1419-1427.

17

12. Trapido, M.; Goi, A. Degradation of nitrophenols with the Fenton reagent. Proc.
Estonian Acad. Sci. Chem. 1999, 48(4), 163-173.

13. Lindsey, M.E.; Tarr, M.A. Quantitation of hydroxyl radical during F enton oxidation
following a single addition of iron and peroxide. Chemosphere 2000, 41 (3), 409-417.

14. Arnold, S.M.; Hickey, W.J.; Harris, R.F. Degradation of atrazine by Fenton’s
reagent: condition Optimization and product quantification. Environ. Sci. T echnol.
1995, 29(8), 2083-2089.

15. Pratap, K.; Lemley, A.T. F enton electrochemical treatment of aqueous atrazine and
metolachlor. J. Agric. Food Chem. 1998, 46 (8), 3285-3291.

16. Dercova, K.; Branislav, V.; Tandlich, R.; Subova, L. Fenton's Type Reaction and
Chemical Pretreatment of PCBs. Chemosphere 1999, 39(15), 2621-2628.

17. Dutta, K.; Mukhopadhyay, S.; Bhattacharjee, S.; Chaudhuri, B. Chemical oxidation of
methylene blue using a Fenton-like reaction. J. Hazard. Mater. 2001, 384(1), 57-71 .

18. Basu, S.; Wei, I.W. Advanced Chemical Oxidation of 2,4,6-Trichlorophenol in
Aqueous Phase by Fenton's Reagent- Part I: Effects of the Amounts of Oxidant and
Catalyst on the Treatment Reaction. Chem. Eng. Commun. 1998, 164, 111-137.

19. Hemer, H.A.; Trosko, J.E.; Masten, S]. The Epigenetic Toxicity of Pyrene and
Related Ozonation Byproducts Containing an Aldehyde Functional Group. Environ.
Sci. T echnol. 2001, 35 (17), 3576-3583.

20. Sandau, C.D.; Ayotte, P.; Dewailly, E.; Duffe, J .; Norstrom, RJ. Analysis of
Hydroxylated Metabolites of PCBs (OH-PCBs) and Other Chlorinated Phenolic
Compounds in Whole Blood from Canadian Inuit. Environ. Health Perspect. 2000,
108(7), 611-616.

21. Campbell, L.M.; Muir, D.C.G.; Whittle, D.M.; Backus, S.; Norstrom, R.J.; Fisk, A.T.
Hydroxylated PCBs and Other Chlorinated Phenolic Compounds in Lake Trout
(Salvelinus namaycush) Blood Plasma from the Great Lakes Region. Environ. Sci.

T echnol. 2003, 3 7 (9), 1720-1725.

22. Machala, M.; Ludék, B.; Lehmler, H.-J.; Pliskova, M.; Majkova, Z.; Kapplova, P.;
Sovadinova, 1.; Vondrééek, J .; Malmberg, T.; Robertson, L.W. Toxicity of
Hydroxylated and Quinoid PCB Metabolites: Inhibition of Gap Junctional
Intercellular Communication and Activation of Aryl Hydrocarbon and Estrogen
Receptors in Hepatic and Mammary Cells. Chem. Res. Toxicol. 2004, 17(3), 340-347.

23. Letcher, R.J.; Klasson-Wehler, E.; Bergman, A. Methyl sulfone and hydroxylated
metabolites of polychlorinated biphenyls. In The handbook of environmental

l8

24.

25.

26.

27.

28.

29.

30.

31.

32.

chemistry Vol. 3 Part K. New Types of persistent halogenated compounds; Paasivirta,
J ., Ed.; Springer-Verlag: Berlin, 2000.

Park, J .-S.; Bergman, A.; Linderhohn, L.; Athanasiadou, M.; Kocan, A.; Petrik, J .;
Drobna, B.; Tmovec, T.; Charles, M.J.; Hertz-Picciotto, I. Placental transfer of
polychlorinated biphenyls, their hydroxylated metabolites and pentachlorophenol in
pregnant women from eastern Slovakia. Chemosphere 2008, 70, 1676-1684.

Wiegel, J .; Zhang, X.; Wu, Q. Anaerobic Dehalogenation of Hydroxylated
Polychlorinated Biphenyls by Desulfitobacterium dehalogenans. Appl. Environ.
Microbiol. 1999, 65 (5), 2217-2221.

Upham, B.L.; Boddy, B.; Xing, X.; Trosko, J .E.; Masten, S.J. Non-Genotoxic Effects
of Selected Pesticides and Their Disinfection By-Products on Gap Junctional
Intercellular Communication. Ozone Sci. Eng. 1997, 19, 351-369.

Kumar, N.M.; Gilula, NB. The Gap Junction Communication Channel. Cell. 1996,
84, 381-388.

Upham, B.L.; Masten, S.J.; Lockwood, B.R.; Trosko, J .E. Nongenotoxic Effects of
Polycyclic Aromatic Hydrocarbons and Their Ozonation By-Products on the
Intercellular Communication of Rat Liver Epithelial Cells. Fundam. Appl. T oxicol.
1994, 23, 470-475.

Trosko, J .E.; Chang, C.C.; Madhukar, B.V.; Klaunig, J .E. Chemical, Oncogene and
growth Factor Inhibition of Gap J unctional Intercellular Communication: An
Integrative Hypothesis of Carcinogenesis. Pathobiology. 1990, 58, 265-278.

Trosko, J. E.; Chang, C.C. Nongenotoxic Mechanisms in Carcinogenesis: Role of
Inhibited Intercellular Communication. Banbury Report 31: Carcinogen Risk
Assessment: New Directions in the Qualitative and Quantitative Aspects. 1988, 139-
169.

Silberhom, E.M.; Glauert, H.P.; Robertson, L.W. Carcinogenicity of Polyhalogenated
Biphenyls: PCBs and PBBs. Crit. Rev. T oxicol. 1990, 20(6), 440-496.

Kang, K.S.; Wilson, M.R.; Hayashi, T.; Chang, C.C.; Trosko, J .E. Inhibition of Gap
Junctional Intercellular Communication in Normal Human Breast Epithelial Cells
afier Treatment with Pesticides, PCBs, and PBBs, Alone or in Mixtures. Environ.
Health Perspect. 1996, 104 (2), 192-200.

19

Chapter 2
Epigenetic Toxicity of Hydroxylated Biphenyls and Hydroxylated Polychlorinated
Biphenyls as Potential Remediation Byproducts
2.1 Introduction

An article (I) based on this chapter was published in Environmental Science and
Technology.

Exposure to polychlorinated biphenyls (PCBs) is known to have numerous human
health effects including reproductive disorders, embryotoxicity, oncogenicity, and
estrogenic endocrine disruption, as well as probable human carcinogenicity (2-4). Within
organisms, PCBs are metabolized into hydroxylated polychlorinated biphenyls (OH-
PCBs) (5). OH-PCBs, which have been detected in human organs, blood, fatty tissue,
and milk, as well as in fish and wildlife, have estrogenic and anti-estrogenic effects, can
disrupt thyroxin and vitamin A transport, and might lead to adverse neurodevelopmental
effects (5-7). Since OH-PCBs are relatively stable in mammalian systems, they are
eliminated mainly by excretion in urine and feces and are detected as residues in the
environment (6). Hydroxylated PCBs can also be formed during Fenton’s oxidation,
which has been found to be an effective method of remediating PCB contaminated soils
and aqueous solutions (8, 9). F enton’s oxidation of PCBs would result in the addition of
a hydroxyl group to nonhalogenated sites and the production of hydroxylated
polychlorinated biphenyls, and further reactions might occur resulting in dechlorination
(8).

The ability to disrupt gap junctional intercellular communication (GJIC) is

indicative of one form of a chemical’s epi genetic toxicity (10). Epigenetic toxicity is but

20

one of three means by which toxic chemicals can alter the functioning of cells: (a)
“genotoxicity” or mutagenesis is when a toxic chemical alters the genetic information of
a cell (the DNA code) or the number or structure of the chromosomes; (b) cytotoxicity is
when a toxic chemical kills a cell by a necrotic mechanism (cell basically dies because of
membrane damage, critical enzymes or proteins are destroyed) or an “apoptotic”
mechanism (a chemical causes the cell to commit suicide (programmed cell death)); or
(c) epigenetic toxicity is when a non-mutagenic and non-cytotoxic chemical alters the
expression of normal genes causing inappropriate “turning on or off” of genes in a cell at
the wrong place at the wrong time (11). The alteration of the expression of genes can
occur at the transcriptional, translational, or posttranslational levels (12). Epigenetic
toxicity can be evaluated using the GJIC assay, a nongenotoxic assay that measures the
level of cell-cell communication. Gap junctional intercellular communication, which is
required for cellular growth control, developmental and differentiation processes,
synchronization of cellular functions, and metabolic regulation, results from the transfer
of ions and small molecules (5. 1200 Da) between cells through membrane channels
called gap junctions (13). It has been suggested by Trosko et al. (14) that the reversible
inhibition of GJIC by endogenous or exogenous tumor promoters is responsible for the
tumor promotion phase of carcinogenesis. Some PCBs have been shown to have
epigenetic toxicity by being capable of disrupting GJIC (15, 16). PCBs with coplanar
conformations appear less likely to inhibit GJIC, whereas decreased coplanarity of the
PCB increases the chances of inhibition of GJIC (1 7—19). Few studies have been
performed to determine the effects of OH-PCBs on GJIC and none have investigated the

OH-PCBs examined in this chapter. Machala et al. (20) observed inhibition of GJIC for a

21

selected group of OH-PCBs, with the strongest inhibition for OH—PCBs with the highest
molecular weight. The majority of the mono- and dihydroxy-PCBS studied inhibited
GJIC in vitro in micromolar concentrations. No cytotoxic effects of the tested
compounds were observed with 30 minutes and 24 hours of exposure at concentrations
up to 100 M. No literature could be found regarding the effects of hydroxylated
biphenyls (OH-BPS) on GJIC.

The objective of this study was to determine whether there is any correlation
between the chemical properties (including structure) of OH-PCBs and OH-BPs and their
observed epi genetic toxicity and cytotoxicity. Six commercially available chemicals
considered to be representative of OH-BPs and OH-PCBs, which are potential byproducts
of the remediation of PCBs with F enton’s reagent, were selected for study. These six
chemicals were 2-biphenylol (2BP), 3-biphenylol (3BP), 2,2'-bipherlyldiol (2,2'BP), 3,3'-
biphenyldiol (3,3'BP), 3-chloro-2-biphenylol (3C2BP), and 4,4'-dichlorO-3-biphenylol
(4,4'DC3BP). The toxicity of these chemicals was evaluated in rat liver epithelial cells
using a nongenotoxic bioassay that determines the in vitro modulation of GJIC as a
measure of the epigenetic toxicity. For each chemical the dose-response, time-response,

time of recovery, and cytotoxicity were investigated.

2.2 Experimental Section
2.2.1 Chemicals

2-Biphenylol, 3-biphenylol, 2,2'-biphenyldiol, 3,3'-biphenyldiol, 3-chloro-2-
biphenylol, and 4,4'—dichloro-3-biphenylol were all purchased from Chem Service Inc.

(West Chester, PA) (Figure 2.1). All chemicals used as toxicants in the study had a

22

H OH H H HO H H H
H H H H H H H H

2-Biphenylol (2BP) 3-Biphenylol (3BP)
H OH HO H HO H H OH
H H H H H H H H
2,2'-Biphenyldiol (2,2’BP) 3,3’-Biphenyldiol (3,3'BP)

Cl OH H H HO H H H
HfiH CIT—Hm
H H H H H H H H

3-Chloro-2-biphenylol (3C2BP) 4,4'-DichlorO-3-biphenylol (4,4'DC3BP)

Figure 2.1 Chemical structures of the six chemicals selected as potential byproducts of
remediation of PCBs with F enton’s reagent and evaluated for toxicity.

23

purity of 95% or greater. Acetonitrile (99.8% purity) was purchased from EM Science
(Gibbstown, NJ). For cell culture, D-medium (Formula No. 78-5470 EG), Fetal Bovine
Serum (F BS), and Gentarnicin were purchased from GIBCO Laboratories (Grand Island,
NY). Lucifer Yellow CH, dilithium salt, was purchased from Molecular Probes Inc.
(Eugene, OR) and ICN Biomedicals Inc. (Aurora, OH). Neutral red dye (3-amino-7-
dimethylamino-Z-methylphenazine hydrochloride) for the cytotoxicity bioassays was
purchased from Sigma Chemical Co. (St. Louis, MO). Acetic acid (99.7 %) was
purchased from EMD Chemicals Inc. (Gibbstown, NJ). Ethanol (95% purity) was
purchased from Aaper Alcohol and Chemical Co. (Shelbyville, KY). Formaldehyde

solution (37%) for the GJIC bioassays was purchased from J .T. Baker (Phillipsburg, NJ).

2.2.2 Methods
2.2.2.1 Toxicological Evaluation

Stock solutions of the purchased OH-BPs and OH-PCBs were prepared in
acetonitrile (ACN). ACN was chosen as the solvent for the stock solutions because it has
minimal effect on GJIC at a final ACN concentration of up to 1.5% in the cell culture
medium as determined by an ACN dose-response test. Since slight inhibition of GJIC
occurs at a final ACN concentration of 2.0% in the cell culture medium (corresponding to

40 pL of ACN), all experiments were conducted at final ACN concentrations of 1.5% or

less (corresponding to 30 uL of ACN or less) (21 ).

24

2.2.2.2 Cell Culture Techniques

WB-F344 rat liver epithelial cells were obtained from Dr. J. W. Grisharn and Dr.
M. S. Tsao of the University of North Carolina (Chapel Hill, NC) (12). This cell line was
selected because it is a diploid, nontumorigenic cell line originating from a strain of rat
that has been used for toxicological/cancer studies of numerous chemicals, thereby
allowing for a source of comparison (12). Since 70% of the chemicals that are
carcinogens are liver carcinogens and the liver is the “first pass” organ for ingested
toxins, liver cells are important for toxicological/cancer studies (22). Furthermore, the
WB-F344 cell line was designed for in vitro assays to match the many in vivo tumor
promotion assays that had been done in rat liver, specifically, in the Fischer 344 rat.

The cell culture techniques performed were similar to those described by Hemer
et al. (12) and Luster-Teasley et al. (23). Cells were cultured in 150 cm2 sterile, treated,
polystyrene cell culture flasks (Corning Inc., Corning, NY) in 25 mL of D-medium
containing 5% Fetal Bovine Serum (FBS) and 0.2% Gentamicin. The cells were
incubated at 37 °C in a water-j acketed IR Autoflow Automatic CO2 incubator (NU AIRE,
Inc., Plymouth, MN) in a humidified atmosphere with 5% CO2 and 95% air. The time
required for cell growth confluency was about two days. The confluent culture was split
and transferred every other day into a new 150 cm2 culture flask with new medium
mixture. In addition, from 150 cm2 flasks of confluent cells, cultures were prepared for
the bioassays in 35 mm diameter, sterile, treated polystyrene cell culture dishes (Corning
Inc., Corning, NY) with 2 mL of D-medium supplemented with 5% FBS. The cultures

for the bioassays were incubated under the same conditions as the aforementioned flasks.

25

2.2.2.3 In Vitro Bioassay for GJIC

The bioassays of GJIC were performed on confluent cell cultures (usually 2 days
of growth) grown in 35 mm diameter culture dishes (as described in the preceding
section). The scrape-loading/dye transfer (SL/DT) procedure for determining the GJIC
was adapted from the method described by El-Fouly et al. (24) and is described in detail
by Hemer et al. (12). A detailed description of the spread of Lucifer yellow dye from the
scrape to neighboring cells can be found in Wilson et al. (25). Chemical treatments,
controls (no dose), and vehicle controls (ACN only) were performed in triplicates. Doses
were applied directly to the dishes of confluent cell cultures. Vehicle controls were
dosed with a volume of ACN corresponding to the largest volume of chemical dose tested
in the treatrrlent dishes. Chemical treatments were performed at noncytotoxic levels as
determined by the neutral red uptake assay (cytotoxicity bioassay) (26). Measurement of
GJIC has to be done at concentrations that do not cause cytotoxicity, since the objective
is to determine if a potential toxicant can bring about its physiological toxic endpoint
without tissue destruction but by noncytotoxic mechanisms.

The specific GJIC bioassays that were performed included dose-response, time-
response, and time of recovery. All culture dishes were examined within 24 hours of the
experiment completion. Each culture dish of cells was digitally photographed such that
the observed scrape spanned the full horizontal width of the picture. A COHU High
Perfomance Color CCD Camera (Cohu, Inc., San Diego, CA) with a magnification of
200x under a Nikon Diaphot-TMD epifluorescence phase-contrast microscope (Nikon
Corp., Japan) illuminated with a Nikon HB-10101AF Super High Pressure Mercury

100W lamp (Nikon Corp., Japan), or a Nikon TE3 00 Eclipse Inverted Microscope (Nikon

26

Corp., Japan) with a Nikon HB-10103AF Super High Pressure Mercury 100W lamp
(Nikon Corp., Japan) was used.

The fluorescence of the Lucifer yellow dye was used to determine the distance the
dye traveled perpendicular to the scrape. This distance of dye travel was indicative of the
level of GJIC within the culture. Quantitative analysis of the distance of dye spread was
performed using NucleOTech GelExpert software (NucleoTech Corp., Hayward, CA).
The distance of dye spread was measured in terms of the area of dye spread, by tracing
manually via free object quantification the area of farthest visible fluorescence. Since the
width Of the photographed section was the same for every culture dish, measuring the
area of the dye spread was equivalent to measuring the distance of dye spread
perpendicular to the scrape. The area of dye spread for each chemical treatment dish was
compared to a control group of cells that were exposed to ACN only (vehicle controls)
under the same assay as the treated cells. For each chemically treated dish, the fraction of
the control was calculated as the area of dye spread in the treated dish divided by the
average area of dye spread in the triplicate set of vehicle control dishes. The results for
each set of chemically treated triplicates were reported as an average fraction of the
control (FOC) :1: standard deviation (SD) determined at the 95% confidence interval (CI).

The level of GJIC in cells exposed to the chemical was assessed by the decrease
in communication of the cells as compared to the vehicle control groups, exposed to
ACN only. A decrease in FOC corresponds directly to a decrease in GJIC (where the
doses are not cytotoxic). Interpretations of GJIC results are consistent with Luster-
Teasley et al. (23) and Hemer et al. (12). Complete communication (100%) between the

cells is identified as a FOC value of 1.0 as seen in the vehicle control. A FOC value

27

greater than 0.9 is difficult to statistically distinguish from the vehicle controls. F 0C
values between 0.9 and 0.5 indicate partial inhibition of GJIC. A FOC value less than or
equal to 0.5 is indicative of a significant amount of inhibition of GJIC, since this would
be representative of communication levels that are 50% or less than the normal
communication levels. FOC values between 0.3 and 0.0 are representative of complete
inhibition of GJIC. A FOC value of 0.3 is usually used to represent complete inhibition,
as it corresponds to the width of a single row of cells with no dye spreading beyond its
boundaries (12). Controls, which received no close of chemical or solvent (ACN), were
performed for each experiment as a means of evaluating a normal level of GJIC and the
overall “health” of the cells. By performing a t-test for each experiment, it was found
that the areas of dye spread for the control dishes (no solvent or chemical) did not vary
significantly from the areas for the vehicle controls (only ACN) at a 95% CI. Therefore,
it could be concluded that the solvent, at the volume tested, was not a significant source
of inhibition in the experiments. Statistical analyses were performed by means of the t-
test and One Way Analysis of Variance (ANOVA) to compare the chemical treatment

results and vehicle control results.

2.2.2.4 In Vitro Bioassay for Cytotoxicity

The procedure for determining cytotoxicity using the neutral red uptake assay
was adapted fi'om the method of Borenfreund and Puemer (26) and the method described
by Weis et al. (2 7). The WB-F344 rat liver epithelial cells were cultured in the same
manner as that used for the GJIC bioassays. Dishes with a confluent growth of cells of

similar density were used for both the dye incubation test and cytotoxicity bioassays.

28

Chemical treatments, controls (no dose) and vehicle controls (ACN only) were performed
in triplicate. A solution of neutral red dye (0.08%) in D-medium supplemented with 5%
FBS was incubated at 37 °C for approximately 2 hours. The neutral red dye solution was
centrifuged at 1200 rpm for 10 minutes and the supernatant was filtered through a 0.22
pm hydrophilic polysulfone membrane filter.

To determine the amount of time necessary for incubation of the dishes with the
neutral red dye solution, a dye incubation test was performed. A set of 8 control dishes
(not chemically treated) were each incubated with 2 mL of the neutral red dye solution at
37 °C in a humidified atmosphere containing 5% C02 and 95% air. At 5, 10, 15, 20, 25,
30, 40, and 60 minutes, a single dish was removed from the incubator, rinsed five times
with Ca2+/Mg2+ PBS (Phosphate Buffered Saline), and 1 mL of neutral red solubilizer (an
aqueous solution of 1% acetic acid, 50% ethanol, and 49% R0. water) was added and
allowed to lyse the cells for 15 minutes at room temperature. The absorbance of the
solubilized dye was measured in cuvettes (polystyrene, optical pathlength of 10 mm) at
540.0 nm on a Beckman DU 7400 Spectrophotometer (Beckman Instruments, Inc.,
Fullerton, CA). A time of dye incubation was selected which gave an absorbance
measurement close to 1.5 at 540.0 nm. If all incubation times yielded an absorbance
greater than 2.0 at 540.0 nm, then the samples of solubilized dye were reanalyzed
following dilution with neutral red solubilizer.

To test the cytotoxicity of a chemical toxicant, after the cells were exposed to the
chemical for the desired incubation time, the cells were rinsed five times with Cari/Mg”
PBS, and 2 mL of the neutral red dye solution was added to each dish. The cells were

then incubated for the predetermined dye incubation time at 37 °C in a humidified

29

atmosphere containing 5% CO2 and 95% air. After incubation, the dishes were rinsed
five times with CaZII/Mg2+ PBS, and 1 mL of neutral red solubilizer was added to each
dish and allowed to lyse the cells for 15 minutes at room temperature. If dilution was
found to be necessary in the dye incubation test, the solubilized dye solutions were
diluted as previously mentioned. The absorbance of the solubilized dye was measured as
aforementioned at 540.0 nm and 630.0 nm. The background absorbance at 630.0 nm was
subtracted from the absorbance measured at 540.0 nm to obtain a true absorbance. For
each chemically treated dish, the fraction of the control was calculated as the true
absorbance in the treated dish divided by the average true absorbance in the triplicate set
of vehicle control dishes. The results for each set of chemically treated triplicates were
reported as an average fraction of the control (FOC) :t standard deviation (SD)
determined at the 95% confidence interval.

A cytotoxicity F 0C of about 1.0 indicates that the neutral red uptake was
approximately equivalent to that of the vehicle control (a noncytotoxic response). A F 0C
value greater than or equal to 0.8 is considered indicative of a noncytotoxic response. A
F 0C value of less than 0.8 indicates that significantly less neutral red dye solution was
retained by the cells than within vehicle control cells; therefore, the chemical is cytotoxic
at that dose (28). Statistical analyses equivalent to those performed for the GJIC

bioassays were performed.

30

2.3 Results and Discussion
2.3.1 30-Minute Cytotoxicity Assay

It is important to use noncytotoxic levels of chemical toxicants in the GJIC
bioassays since if a cytotoxic dose were used, it would be impossible to distinguish
between decreased intercellular communication due to cell death and decreased
intercellular communication due to inhibition Of GJIC by closure or constriction of the
gap junctions. The concept behind the neutral red uptake assay is that viable cells will
incorporate the dye by active transport, whereas nonviable cells will not. A change in the
number of cells or their physiological state will result in a direct change in the amount of
dye the cells incorporate and, hence, the degree of cytotoxicity indicated. For the 30
minute cytotoxicity experiments, confluent growths of cells of similar density to those
used in the bioassays for GJIC were exposed to varying chemical doses and allowed to
incubate for 30 minutes before assaying for cytotoxicity. As shown in Figure 2.2, each
chemical was tested at a dose range of 0 to 300 uM.

Four of the chemicals examined (2BP, 3BP, 2,2'BP, and 3C2BP) were not
cytotoxic within the dose range of 0 to 300 uM. 3C2BP produced FOC values that

oscillated around 0.8 (range 0.70 :t 0.03 to 0.89 i 0.17) for doses between 200 and 300

uM. Typically when cytotoxicity occurs, the FOC will continue to decrease following an
initial downward trend, rather than oscillate about a value. In addition, using one way
analysis of variance to compare the cytotoxicity results within this region of oscillation
indicated that the oscillation about the FOC of 0.8 is not statistically significant. One

could consequently assume that the results for 3C2BP are not indicative of cytotoxicity.

31

Figure 2.2 Cytotoxicity results using the neutral red uptake assay for each of the six
selected potential remediation byproducts. Cell cultures were exposed to the chemicals
for 30 minutes. Each chemical was tested for a dose range of 0 to 300 uM. Each data
point is representative of the results for a set of chemically treated triplicates reported as
an average FOC i standard deviation determined at the 95% confidence interval.

32

 

 

 

 

 

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3,3'BP and 4,4’DC3BP were the only two chemicals found to be cytotoxic.
Between doses of 0 to 280 llM, no cytotoxicity was observed for 3,3'BP. At a dose of
300 uM; however, 3,3'BP was found to be cytotoxic (FOC = 0.69 i 0.03). Cytotoxicity
was observed for 4,4'DC3BP at doses greater than 40 uM. The F 0C rapidly declined as

doses of 4,4'DC3BP increased above 40 uM.

2.3.2 Dose-Response Bioassay

For the dose-response experiments, confluent cells were exposed to varying
chemical doses and allowed to incubate for 30 minutes before assaying for GJIC. Each
chemical was tested over a dose range Of 0 to 300 uM. Chemical doses greater than 300
uM were not tested in order to ensure that ACN final concentrations were not greater
than 1.5 % (discussed in detail in the Experimental Section) and that doses were below
the solubility of the chemical in the cell culture media. Figure 2.3 presents a comparison
of the dose-response curves for the six chemicals tested.

The maximum levels of inhibition occurred for 2,2'BP at 300 uM (FOC = 0.33 i
0.05) and 4,4'DC3BP at 40 uM (FOC = 0.46 i 0.03). 2,2'BP and 4,4'DC3BP, the two
chemicals that exhibited a steep decline in GJIC with an increase in dose, attained a
significant amount of inhibition of GJIC (FOC S 0.5). 2,2'BP was the only chemical to
achieve complete inhibition (this occurs at 300 uM). Since 300 uM was not found to be
a cytotoxic dose for 2,2’BP, the complete inhibition of GJIC observed is not likely to be
due to cytotoxicity, but rather a consequence of chemical inhibition. Although a

continued decline in communication was Observed for 4,4'DC3BP at doses greater than

34

Figure 2.3 Dose-response results for each of the six selected potential remediation
byproducts. Cell cultures were exposed to the chemicals for 30 minutes. Each chemical
was tested for a dose range of 0 to 300 uM. Each data point is representative of the

results for a set of chemically treated triplicates reported as an average FOC 2|: standard
deviation determined at the 95% confidence interval.

35

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40 M, this lack of communication can be attributed to cytotoxicity rather than inhibition

of GJIC.

No dose-response data were obtained for 4,4'DC3BP at doses above 100 uM,
since at concentrations of 150 M and greater it became impossible to quantitate a level
of dye spread. When observed under visible light (phase contrast) at these higher doses,
detachment of cells fiom the surface of the dish became apparent as sections of missing
cells in the otherwise confluent layer. When observed under ultraviolet (UV) light at
these same doses, there was no apparent front of dye spread, but rather a fluorescing of
random or remaining cells on the dish. Figure 2.4 depicts phase contrast and UV
epifluorescent images, which compare 0 and 250 p.M doses of.4,4'DC3BP. The
detachment of cells from the surface of the dish might be attributed to the chemical’s
effect on the cell adhesion proteins, which adhere the cells to the treated dish surface,
resulting in subsequent cell death. Fluorescing of random or remaining cells on the dish
is indicative of perforation of the cell membranes by chemical damage, allowing the dye
to enter the cell freely rather than passing through the gap junctions from neighboring
cells. As shown in Figure 2.4B and D, the presence of isolated fluorescing cells, lacking
direct contact to neighboring cells, is further indication of perforation of the cell
membranes, since dye could not reach these cells via passage through gap junctions.
Both the loss of adhesion of the cells and perforation of the cell membranes are consistent
with 30 minute cytotoxicity results.

2BP, 3BP, 3,3'BP, and 3CZBP exhibited a gradual decline in GJIC with an
increase in dose. Only partial inhibition of GJIC (FOC between 0.9 and 0.5) was

observed for these chemicals. In terms of increasing maximum inhibition at noncytotoxic

37

Figure 2.4 Phase contrast (visible light) and UV epifluorescent photomicrographs at
200x magnification comparing 0 and 250 pM doses of 4,4'DC3BP. A treatment of 0 14M
4,4'DC3BP for 30 minutes was a vehicle control treatment with 25 uL of ACN for 30
minutes. (A) Phase contrast and (C) UV epifluorescent photomicrographs of cell cultures
treated with 0 uM 4,4'DC3BP for 30 minutes indicate a confluent layer of healthy cells
with complete communication. (B) Phase contrast and (D) UV epifluorescent
photomicrographs of cell cultures treated with 250 uM 4,4'DC3BP for 30 minutes
indicate detachment of cells fiom the cell monolayer and a fluorescing of the remaining
cells.

38

 

39

levels, the order of these chemicals was 3,3'BP (FOC = 0.71 i 0.03 at 250 11M), 3BP
(FOC = 0.63 i 0.02 at 300 uM), 3C2BP (FOC = 0.61 i 0.03 at 300 uM), and 2BP (FOC
= 0.56 i 0.02 at 300 uM). On the basis Of the results of the 30 minute cytotoxicity assay,
the reduction in communication observed at 300 uM for 3,3'BP is a consequence of

cytotoxicity rather than chemical inhibition of GJIC.

2.3.3 24-Hour Cytotoxicity Assay

A 24 hour cytotoxicity assay was performed to aid in the selection of the dose to
be used for the time-response experiment. To determine whether a reduction in
intercellular communication at 24 hours of toxicant exposure is due to chemical
inhibition by closure or constriction of the gap junctions rather than cytotoxicity, it is
essential that the dose selected is not cytotoxic at 24 hours of toxicant exposure. The
only variation of the 24 hour cytotoxicity assay from the 30 minute cytotoxicity assay
was the 24 hour exposure period. In addition, the cells were evaluated visually (under a
microscope) after 24 hours for indications of cell stress or detachment of cells from the
dish surface.

Table 2.1 presents the 24 hour cytotoxicity results for the selected time-response
dose for each chemical evaluated. None of the six chemicals studied were cytotoxic after
24 hours of toxicant exposure at the doses used for the time-response experiments.
Hence, it can be assumed that any inhibition observed at these doses for up to 24 hours of
toxicant exposure could be attributed to chemical inhibition by closure or constriction of

the gap junctions.

40

Table 2.1 24 Hour Cytotoxicity at the Doses Used for the Time-Response Bioassays

 

 

 

Time-Response Dose 24 hr Neutral Red Dye Uptake
Compound

QIM) (FOC :1: SD)
2-biphenylol 230 1.04 i 0.10
3-biphenylol 150 0.93 i 0.07
2,2’-biphenyldiol 250 0.85 i 0.11
3,3'-biphenyldiol 250 1.13 i 0.16
3-chloro-2-biphenylol 100 1.25 i 0.11
4,4'-dichloro-3-biphenylol 40 0.93 i 0.03

 

 

 

41

 

2.3.4 Time-Response Bioassay

The time-response assay was used to determine the effect of toxicant exposure
time on intercellular communication. For the time-response experiments, confluent
cultures of cells were exposed to a fixed dose of toxicant for varying amounts of time
ranging from 0 to 24 hours (1440 minutes) followed by GJIC bioassays. The dose for the
time-response experiment for each chemical was selected as the highest dose that is not
cytotoxic at 24 hours of toxicant exposure, but causes a substantial amount of inhibition
at 30 minutes of toxicant exposure. Figure 2.5 is a comparison of the time-response
curves for the six chemicals tested. The dose selected for the time-response experiment
for each chemical is indicated in the legend of this figure.

2BP and 3BP both rapidly inhibited GJIC. Within 30 minutes of toxicant
exposure, GJIC was reduced to a FOC of 0.60 i 0.04 for 2BP and a FOC of 0.67 i 0.01
for 3BP. This rapid rate of inhibition suggests that inhibition was a consequence of the
parent compound (2BP or 3BP), rather than a metabolite, which would require more time
to form. 2BP was the only chemical to achieve almost complete inhibition (F OC = 0.36
$0.04) within 24 hours of toxicant exposure. A maximum level of inhibition for 3BP
(F 0C = 0.48 320.03) was attained at 24 hours of toxicant exposure. For BBP, between 5
minutes and 30 minutes, a slight increase and then decrease of FOC was observed, which
might indicate a brief period of adaptation followed by resumed inhibition.

The inhibition of GJIC by 2,2'BP and 4,4'DC3BP occurred rapidly. 2,2'BP
achieved maximum inhibition at 45 minutes (FOC = 0.55 i 0.02), while 4,4'DC3BP
achieved a FOC of 0.52 3: 0.02 at 10 minutes and then maintained an average FOC of

0.50 until 720 minutes (12 hours). A partial recovery of communication without removal

42

Figure 2.5 Time-response results for each of the six selected potential remediation
byproducts. The legend in the figure indicates the dose used for each of the chemicals.
Time of exposure varied from 0 minutes to 1440 minutes (24 hours). Each data point is
representative of the results for a set of chemically treated triplicates reported as an
average FOC i standard deviation determined at the 95% confidence interval. Data
points after the break correspond to 120 minutes, 240 minutes, 360 minutes, 480 minutes,
600 minutes, 720 minutes, and 1440 minutes of chemical exposure, respectively.

43

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of the chemical was exhibited for both chemicals. For 2,2'BP, partial recovery occurred
between 45 and 720 minutes (a period of 675 minutes) with a FOC of 0.81 i 0.03 as the
maximum recovery. This level of maximum recovery was maintained through 1440
minutes. For 4,4'DC3BP, partial recovery occurred between 720 minutes and 1440
minutes (a period of 720 minutes) with a FOC of 0.68 i 0.01 as the maximum recovery.
Although GJIC was used as a screening “biomarker” and not for the purpose of studying
biochemical mechanisms, one possible speculative explanation for this partial recovery
without removal of the chemical might be the activation of an autoregulatory pathway.
In this case, the chemical would activate a pathway that would act to inhibit GJIC and
then, in an autoregulatory fashion, another pathway would be activated which would
reestablish GJIC. This partial recovery might also be attributed to the cell’s ability to
adapt to the change in conditions (such as cell homeostasis) due to toxicant exposure that
resulted in the initial inhibition. Finally, partial recovery without the removal of the
chemical might be a consequence of the cells metabolizing the chemical into less toxic
metabolites.

The inhibition of GJIC by 3,3'BP and 3C2BP occurred slowly. Within 30
minutes of toxicant exposure, 3,3'BP achieved a FOC of 0.79 i 0.03, while 3C2BP
achieved a FOC of 0.81 i 0.03. The decline in communication for 3,3'BP continued to a
maximum level of inhibition of a FOC of 0.51 i 0.02 at 24 hours (1440 minutes). This
slow rate of inhibition observed for 3,3'BP might be a consequence of the cells slowly
metabolizing this chemical into a toxic metabolite. No significant level of inhibition was

observed for 3C2BP during the 24 hours of exposure time. The maximum level of

45

inhibition for 3C2BP was only a FOC of 0.68 i 0.03 at 24 hours, which is equivalent to

the amount of inhibition obtained for 3BP at 30 minutes.

2.3.5 Time of Recovery Bioassay

The time of recovery assay evaluated the ability of toxicant exposed cells to
recover complete communication following the removal of the toxicant and replacement
with new medium. From the results of the time-response bioassay, an incubation time for
each chemical was selected that resulted in either a significant amount of inhibition or an
amount of inhibition of interest. For each chemical, cells were exposed to a fixed dose of
toxicant identical to that selected for the time-response experiment and were incubated
for the selected incubation time. Following incubation, the cells were rinsed with
Ca2+/Mg2+ PBS, new medium was added, and the cells were returned to the incubator for
varying amounts of recovery time followed by GJIC bioassays. Figure 2.6 is a
comparison of the time of recovery curves for five of the chemicals tested. Since no
significant level of inhibition was observed for 3C2BP during the time-response
experiment, no time of recovery experiment was performed for this chemical. The dose
and incubation time selected for the time of recovery experiment for each chemical is
indicated in the legend of this figure. Complete recovery is attained at a FOC value of
0.97 and greater.

For all of the chemicals tested, cell cultures completely recovered communication
following removal of the toxicant from the cell cultures. The times required to achieve

complete recovery after removal of 2BP, 3BP, 2,2'BP, 3,3'BP, and 4,4'DC3BP were 240

minutes, 120 minutes, 240 minutes, 360 minutes, and 480 minutes, respectively. Trosko

46

Figure 2.6 Time of recovery results for five of the six selected potential remediation
byproducts. Since no significant level of inhibition was observed in the time-response
results for 3C2BP, no time of recovery experiment was performed for this chemical. The
legend in the figure indicates the dose and incubation time selected for the time of
recovery experiment for each chemical. Each data point is representative of the results
for a set of chemically treated triplicates reported as an average F OC at standard deviation
determined at the 95% confidence interval. Data points after the break correspond to 60
minutes, 120 minutes, 240 minutes, 360 minutes, 480 minutes, and 600 minutes of
recovery, respectively.

47

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et al. (13) hypothesized that most, if not all, tumor promoting chemicals reversibly inhibit
GJIC. One could therefore hypothesize that the reversible inhibition of GJIC observed
for these five chemicals in vitro is predictive of their potential to be tumor promoters in
vivo as well as other disease endpoints (29). In addition, based on similar results from
time of recovery experiments performed with fluoranthene and pyrene, Upham et al. (21)
suggested that the ability of cells to completely recover fi'om a substantial level of
inhibition indicated that the inhibition was probably not at the transcriptional or gene
level but was rather a consequence of a post-translational biochemical change.
Furthermore, the complete recovery of communication indicates indirectly that the
chemicals tested were not cytotoxic at the inhibitory doses used for the time of recovery
experiment (12).

A cyclic or oscillatory behavior of GJIC FOC was observed during the first 30
minutes of recovery time for all the chemicals tested. This phenomenon might be
indicative of a period of adaptation following the removal of the chemical and might
entail the re-opening of existing, closed channels followed by the synthesis of new
channels. At recovery times 60 minutes and longer, there was a smooth increase in GJIC
until complete recovery was achieved.

As presented in the time-response section, 2,2'BP and 4,4'DC3BP both exhibited
partial recovery without removal of the chemical. In the time-response experiment, in
which 2,2'BP was not removed from the cell culture, partial recovery (FOC = 0.81 i
0.03) occurred 675 minutes (11 hours 15 minutes) after inhibition reached a maximum.
In the time of recovery experiment, however, complete recovery occurred 240 minutes (4

hours) after removal of 2,2'BP. Similarly, in the time-response experiment, in which

49

4,4'DC3BP was not removed from the cell culture, partial recovery (FOC = 0.68 i- 0.01)
occurred 720 minutes (12 hours) after inhibition reached a maximum. In the time of
recovery experiment, however, complete recovery occurred 480 minutes (8 hours) after
removal of the chemical. For both chemicals, there was a greater extent and faster rate of
recovery of communication after removal of the chemical than without removal of the

chemical for doses studied.

2.3.6 Estimation of Octanol/Water Partition Coefficients and Solubility in Water

To correlate the observed toxicity to the chemical properties, the octanol/water
partition coefficient (Kow) and the solubility in water (S) were estimated for each
chemical studied. Kow values have been shown to be useful in associating structural
changes in drug chemicals with observed changes in various biological, biochemical, or
toxic effects (30). Kow values were estimated using Leo’s Fragment Constant Method
(30). The estimations of Kow for each of the chemicals studied were based on the log
Kow for 2,4,5,2',5'- PCB (31). The solubility in water for each chemical was estimated
using a regression equation for the estimation of S (Eqn. 1) representative of aromatics
and chlorinated hydrocarbons (3 0).

log S = - 1.37 log Kow + 7.26 (Eqn.1)

Table 2.2 presents the estimated log Kow and S values for the chemicals studied, where S
is represented as units of mol/L.

On the basis of the estimations of log Kow and S, it appears that OH-BPs with
only one OH group (2BP and 3BP) were more hydrophobic than OH-BPs with two OH

groups (2,2’BP and 3,3'BP). This is consistent with the observation that functional

50

Table 2.2 Estimated Octanol/Water Partition Coefficients and Water Solubility

 

 

 

 

 

Compound log Kow (moSl/L)
2-biphenylol 1 .89 0.047
3-biphcnylol 1 .89 0.047
2,2'-biphenyldiol 1.22 0.388
3,3'-biphenyldiol 1 .22 0.3 88
3-chlorO-2-biphenylol 2.60 4.99x 10'3
4,4'-dichloro-3-biphenylol 3.31 5.31x10'4

 

 

51

groups containing oxygen tend to be involved in polar and hydro gen—bonding interactions
and act to pull the rest of the molecule in which they are contained into the polar aqueous
phase (32). OH-PCBs appear to be more hydrophobic than OH-BPs. Within the
category of OH-PCBs, solubility in water appears to decrease with an increase in the
number of chlorine functional groups.

4,4'DC3BP exhibited the highest estimated log Kow and lowest estimated S. The
aforementioned cytotoxicity and GJIC inhibitory effects observed for 4,4'DC3BP might

be, although not exclusively, a consequence of the lipophilic nature of this chemical,

which would permit diffusion across the phospholipid bilayer of the cellular membrane.

2.3.7 Structure-Toxicity Relationships

Structure might be influential in determining the toxicity observed for the OH-
BPs and OH-PCBs studied. Biphenyls are remarkable in that a rotational degree of
freedom is present in the phenyl-phenyl linkage that is absent in most polycyclic aromatic
compounds (33). Since chlorines are bulky atoms, it has been shown that substitution of
chlorines at various positions, especially at ortho positions, of the biphenyl can result in
constraints on the rotational freedom of the phenyl-phenyl linkage (34). PCBs lacking
ortho-substituted chlorine atoms may have a coplanar conformation, whereas an
increased number of chloro substituents in ortho positions has been shown to result in
decreased coplanarity (19). Similarly, due to their bulky nature, substitutions by
hydroxyl fimctional groups on a biphenyl can be considered to impart constraints on the
rotational fieedom of the phenyl-phenyl linkage. The rotational degree of freedom in the

biphenyl molecule can also be influenced by attractive forces or bonding between

52

functional groups. Dr. H. Rosenkranz, using a modeling system based on multiple
chemical attributes, has come to the conclusion that GJIC is a very reliable biomarker for
chemical terato gens, tumor promoters, irnmunotoxicants, reproductive toxicants, and
neurotoxicants (29).

Although 2,2'BP and 3,3'BP both possess two hydroxyl functional groups, 2,2'BP
was found to be more inhibitory to GJIC. The dissimilarity between the toxicity results
for these two chemicals might be explained by their structural differences. For 2,2’BP,
the presence of hydroxyl groups at two ortho positions of adjacent aromatic rings would
be expected to result in steric hindrance, thereby increasing the average dihedral (or
twist) angle between the aromatic rings. In addition, significant hydrogen bonding would
be expected to occur between the hydroxyl functionalities on the adjacent aromatic rings
of 2,2'BP. This intramolecular hydrogen bonding causes the protic hydrogen of the free
hydroxyl group to become more favorable for deprotonation, in comparison to the
hydroxyl groups of 3,3'BP (35). The recovery of GJIC without the removal of the
chemical exhibited in the time-response bioassay results for 2,2'BP might be a
consequence of metabolism, in which such deprotonation results in a less toxic
metabolite. In the case of 2,2'BP conformation, it can be expected that steric congestion
factors will predominate over hydrogen bonding, resulting in greater noncoplanarity than
3,3'BP, which lacks steric hindrance and hydrogen bonding. The more noncoplanar
conformation of 2,2'BP might explain the more inhibitory behavior of 2,2'BP to GJIC in
comparison to 3,3'BP. Hemming et al. speculated that a decreased coplanarity increases

the affinity for a cellular target site that is critical for inhibition of GJIC (I9).

53

2BP and 3BP both possess one hydroxyl group; however, 2BP was observed to be
slightly more inhibitory to GJIC. In the structure of 2BP, the introduction of an ortho-
hydroxyl group to the biphenyl brings the oxygen and hydrogen atoms within the van der
Waals radii (36). Similar to 2,2'BP, steric hindrance would occur within the ZBP
molecule due to the presence of a bulky hydroxyl group. In the case of ZBP
conformation, it can be expected that steric congestion factors will predominate over any
van der Waals forces of attraction, increasing the average dihedral angle between the
aromatic rings and increasing the noncoplanarity of the molecule over that of 3BP. The
more noncoplanar conformation of 2BP might explain the more inhibitory behavior of
2BP to GJIC in comparison to 3BP.

For 3C2BP, as previously mentioned for 2BP, the presence of an ortho-hydroxyl
group on the biphenyl structure would be expected to bring the oxygen and hydrogen
atoms within the van der Waals radii. One would expect the same steric hindrance in the
3C2BP molecule as was observed in 2BP, increasing the average dihedral angle between
the aromatic rings and increasing the noncoplanarity of the molecule. The observation
that 3C2BP was less inhibitory to GJIC than 2BP might be explained by the presence of
the chlorine fimctional group, which might act to attenuate the toxic effect of the ortho-
hydroxyl group.

Since for 4,4'DC3BP there is an absence of any ortho-hydroxyl groups, no steric
hindrance, hydrogen bonding, or attractive forces are expected. The conformation of the
molecule is not expected to be as noncoplanar as 2,2’BP, 2BP, and 3CZBP. Introduction
Of meta-chloro substituents into a 4,4'-dichlorobiphenyl molecule has been shown to have

significant effects on the resultant toxicity of the compound (37). In the case of

54

4,4'DC3BP, the introduction of a meta-hydroxyl substituent into a 4,4'-dichlorobiphenyl
molecule might have a similar toxic effect as illustrated by the observed cytotoxicity and
inhibition of GJIC. One possible explanation for the significant amount of inhibition of
GJIC observed for 4,4'DC3BP is hindrance of the rotation of the biphenyl rings by the
hydroxyl group, despite the absence of ortho-substitution, resulting in a noncoplanar
(twisted) conformation of the chemical structure (38). Alternatively, the hydroxyl group
may create a bay-like region on the molecule, which has been shown to result in

inhibition of GJIC and activation of MAPK (mitogen-activated protein kinase) (38, 39).

2.4 Conclusions

2-Biphenylol, 3—biphenylol, 2,2'-biphenyldiol, 3,3'-biphenyldiol, 3-chlorO-2-
biphenylol, and 4,4'-dichloro-3-biphenylol were evaluated using the scrape-loading/dye
transfer (SL/DT) technique to determine the in vitro modulation of gap junctional
intercellular communication (GJIC) in a normal rat liver epithelial cell line as a measure
of the epigenetic toxicity. Cytotoxicity was determined using the neutral red uptake
assay. A dose range of 0 to 300 uM was examined. Only 3,3'-biphenyldiol and 4,4'-
dichloro-3-biphenylol induced cytotoxicity within the tested dose ranges. Noncytotoxic
doses were selected for evaluation of epigenetic toxicity. 4,4’-Dichloro-3-biphenylol was
most inhibitory to GJIC at the lowest dose. The cytotoxicity and GJIC inhibitory effects
Observed for 4,4'-dichlorO-3-biphenylol might be, although not exclusively, a
consequence of the lipophilic nature of this chemical. Alternative explanations for the
significant amount of inhibition of GJIC observed for 4,4'-dichloro-3-biphenylol are (1)

hinderance of the rotation of the biphenyl rings by the hydroxyl group resulting in a

55

noncOplanar conformation and (2) creation of a bay-like region on the molecule by the
hydroxyl grOUp. 3-Chloro-2-biphenylol was least inhibitory to GJIC. 3-Chloro-2-
biphenylol was less inhibitory to GJIC than 2-biphenylol because of the presence of the
chlorine fimctional group, which appears to attenuate the toxic effect of the ortho-
hydroxyl group. Although cells were capable of complete recovery of GJIC after
removal of each of the chemicals, only with 2,2'-biphenyldiol and 4,4'-dichloro-3-
biphenylol did the cells demonstrate partial recovery without removal of the chemical.
The more noncoplanar conformation of 2,2'-biphenyldiol and 2-biphenylol might explain
their more inhibitory behavior in comparison to 3,3'-biphenyldiol and 3-biphenylol,

respectively.

56

2.5 References

1.

Satoh, A.Y.; Trosko, J .E.; Masten, S.J. Epigenetic Toxicity of Hydroxylated
Biphenyls and Hydroxylated Polychlorinated Biphenyls on Normal Rat Liver
Epithelial Cells. Environ. Sci. T echnol. 2003, 3 7 (12), 2727-2733.

Danse, I.R.; Jaeger, R.J.; Kava, R.; Kroger, M.; London, W.M.; Lu, F.C.; Maickel,
R.P.; McKetta, J .J .; Newell, G.W.; Shindell, S.; Stare, F.J.; Whelan, E. M. Review:
Position Paper of the American Council on Science and Health: Public Health
Concerns about Environmental Polychlorinated Biphenyls (PCBs). Ecotoxicol.
Environ. Saf 1997, 38, 71-84.

Soto, A.M.; Sonnenschein, C.; Chung, K.L.; Fernandez, M.F.; Olea, N.; Serrano, F0.
The E-SCREEN Assay as a Tool to Identify Estrogens: An Update on Estrogenic
Environmental Pollutants Environ. Health Perspect. 1995, 103 (7), 113-122.

US. Environmental Protection Agency, Environmental Criteria and Assessment
Ofiice, Office of Health and Environmental Assessment. Integrated Risk Information
System (IRIS) on PCB; Office of Research and Development: Cincinnati, OH, 1994.

Sandau, C.D.; Ayotte, P.; Dewailly, E.; Duffe, J .; Norstrom, R.J. Analysis of
Hydroxylated Metabolites of PCBs (OH-PCBs) and Other Chlorinated Phenolic
Compounds in Whole Blood from Canadian Inuit. Environ. Health Perspect. 2000,
108(7), 611-616.

Wiegel, J .; Zhang, X.; Wu, Q. Anaerobic Dehalogenation of Hydroxylated
Polychlorinated Biphenyls by Desulfitobacterium dehalogenans. Appl. Environ.
Microbiol. 1999, 65 (5), 2217-2221.

Sandau, C.D.; Ayotte, P.; Dewailly, E.; Duffe, J. ; Norstrom, R.J. Pentachlorophenol
and Hydroxylated Polychlorinated Biphenyl Metabolites in Umbilical Cord Plasma of
Neonates from Coastal Populations in Quebec. Environ. Health Perspect. 2002, I 10
(4), 41 1-417.

Sedlak, D.L.; Andren, A.W. Aqueous-Phase Oxidation of Polychlorinated Biphenyls
by Hydroxyl Radicals. Environ. Sci. Technol. 1991, 25 (8), 1419-1427.

Sato, 0; Leung, S.W.; Bell, H.; Burkett, W.A.; Watts, R.J. Decomposition of
Perchloroethylene and Polychlorinated Biphenyls with Fenton's Reagent. In
Emerging Technologies in Hazardous Waste Management 111; Teddler, D. W.,
Pohland, F. G., Eds; ACS Symposium Series 518; American Chemical Society:
Atlanta, GA, 1991; pp 343-356.

10. Trosko, J .E.; Chang, C.C.; Upham, B.; Wilson, M. Epigenetic toxicology as toxicant-

induced changes in intracellular signalling leading to altered gap junctional
intercellular communication. T oxicol. Lett. 1998, 1 02-1 03, 71-78.

57

11. Upham, B.L.; Boddy, B.; Xing, X.; Trosko, J .E.; Masten, SJ. Non-Genotoxic Efiects
of Selected Pesticides and Their Disinfection By-Products on Gap Junctional
Intercellular Communication. Ozone Sci. Eng. 1997, 19, 351-369.

12. Hemer, H.A.; Trosko, J .E.; Masten, S.J. The Epigenetic Toxicity of Pyrene and
Related Ozonation Byproducts Containing an Aldehyde Functional Group. Environ.
Sci. T echnol. 2001, 35 (17), 3576-3583.

13. Trosko, J .E.; Madhukar, B.V.; Chang, C.C. Endogenous and Exogenous Modulation
of Gap Junctional Intercellular Communication: Toxicological and Pharmacological
hnplications. Life Sci. 1993, 53, 1-19.

14. Trosko, J .E.; Ruch, R.J. Cell-Cell Communication in Carcinogenesis. Frontiers
Biosci. 1998, 3, 208-236.

15. Safe, S. Polychlorinated biphenyls (PCBs): Mutagenicity and Carcinogenicity. Mutat.
Res. 1989, 220, 31-47.

16. Tsushimoto, G.; Asano, S.; Trosko, J. E.; Chang, C.C. Chapter 18: Inhibition of
Intercellular Communication By Various Congeners of Polybrominated Biphenyl and
Polychlorinated Biphenyl. In PCBs: Human and Environmental Hazards; D'Itri, F .
M., Kamrin, M.A., Eds; Butterworth Publ.: Boston, MA, 1983; pp 241-252.

17. Silberhom, E.M.; Glauert, H.P.; Robertson, L.W. Carcinogenicity of Polyhalogenated
Biphenyls: PCBs and PBBs. Crit. Rev. T oxicol. 1990, 20(6), 440-496.

18. Kang, K.S.; Wilson, M.R.; Hayashi, T.; Chang, C.C.; Trosko, J .E. Inhibition of Gap
Junctional Intercellular Communication in Normal Human Breast Epithelial Cells
after Treatment with Pesticides, PCBs, and PBBs, Alone or in Mixtures. Environ.
Health Perspect. 1996, 104 (2), 192-200.

19. Hemming, H.; Wamgard, L.; Ahlborg, U.G. Inhibition of Dye Transfer in Rat Liver
WB Cell Culture by Polychlorinated Biphenyls. Pharmacol. T oxicol. 1991, 69(6),
416-420.

20. Machala, M.; Ludek, B.; Lehmler, H.-J.; Pliskova, M.; Majkova, Z.; Kapplova, P.;
Sovadinova, 1.; Vondrééek, J .; Malmberg, T.; Robertson, L.W. Toxicity of
Hydroxylated and Quinoid PCB Metabolites: Inhibition of Gap Junctional
Intercellular Communication and Activation of Aryl Hydrocarbon and Estrogen
Receptors in Hepatic and Mammary Cells. Chem. Res. Toxicol. 2004, 1 7(3), 340-
347.

21. Upham, B.L.; Masten, S.J.; Lockwood, B.R.; Trosko, J .E. Nongenotoxic Efiects of
Polycyclic Aromatic Hydrocarbons and Their Ozonation By-Products on the

58

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

Intercellular Communication of Rat Liver Epithelial Cells. F undam. Appl. Toxicol.
1994, 23, 470-475.

Trosko, J .E. Michigan State University, East Lansing, MI. Personal Communication,
August 2003.

Luster-Teasley, S.L.; Yao, J .J.; Hemer, H.A.; Trosko, J .E.; Masten, S.J. Ozonation of
Chrysene: Evaluation of Byproduct Mixtures and Identification of Toxic Constituent.
Environ. Sci. Technol. 2002, 36 (5), 869-876.

El-Fouly, M.H.; Trosko, J .E.; Chang, C.C. Scrape-Loading and Dye Transfer: A rapid
and simple technique to study gap junctional intercellular communication. Exp. Cell
Res. 1987, 168, 422-430.

Wilson, M.R.; Close, T.W.; Trosko, J .E. Cell Population Dynamics (Apoptosis,
Mitosis, and Cell-Cell Communication) during Disruption of Homeostasis. Exp. Cell
Res. 2000, 254, 257-268.

Borenfreund, E.; Puemer, J .A. Toxicity Determined In Vitro By Morphological
Alterations And Neutral Red Absorption. T oxicol. Lett. 1985, 24, 119-124.

Weis, L.M.; Rummel, A.M.; Masten, S.J.; Trosko, J .E.; Upham, B.L. Bay or Baylike
Regions of Polycyclic Aromatic Hydrocarbons Were Potent Inhibitors of Gap
Junctional Intercellular Communication. Environ. Health Perspect. 1998, 106 (l), 17-
22.

Herner, H.A. Ph.D. Dissertation, Michigan State University, East Lansing, MI, 1999.

Rosenkranz, H.S.; Pollack, N.; Cunningham, A.R. Exploring the relationship between
the inhibition of gap junctional intercellular communication and other biological
phenomena. Carcinogenesis 2000, 21 (5), 1007-1011.

Lyman, W.J.; Reehl, W.F.; Rosenblatt, D.H. Handbook of Chemical Property
Estimation Methods: Environmental Behavior of Organic Compounds; McGraw-Hill
Book Company: New York, NY, 1982.

Chiou, C.T.; Freed, V.H.; Schmedding, D.W.; Kohnert, R.L. Partition Coefficient and
Bioaccumulation of Selected Organic Chemicals. Environ. Sci. Technol. 1977, 11 (5),
475-478.

Schwarzenbach, R.P.; Gschwend, P.M.; Irnboden, D.M. Environmental Organic
Chemistry; John Wiley & Sons, Inc.: New York, NY, 1993.

Barich, D.H.; Pugrnire, R.J.; Grant, D.M.; Iuliucci, R.J. Investigation of the Structural

Conformation of Biphenyl by Solid State 13 C NMR and Quantum Chemical NMR
Shift Calculations. J Phys. Chem. A 2001, 105, 6780-6784.

59

34.

35.

36.

37.

38.

39.

McFarland, V.A.; Clarke, J .U. Environmental Occurrence, Abundance, and Potential
Toxicity of Polychlorinated Biphenyl Congeners: Considerations for a Congener-
Specific Analysis. Environ. Health Perspect. 1989, 81, 225-239.

Mohanty, J .; Pal, H.; Sapre, A.V. Photophysical Properties of 2,2'- and 4,4'-
Biphenyldiols. Bull. Chem. Soc. Jpn. 1999, 72 (10), 2193-2202.

Oki, M.; Iwamura, H. Steric Effects on the O—H-"it Interaction in 2-
Hydroxybiphenyl. J. Am. Chem. Soc. 1967, 89(3), 576-579.

Safe, 3.; Bandiera, S.; Sawyer, T.; Robertson, L.; Safe, L.; Parkinson, A.; Thomas,
P.E.; Ryan, D.E.; Reik, L.M.; Levin, W.; Denomme, M.A.; Fujita, T. PCBs:
Structure-Function Relationships and Mechanism of Action. Environ. Health
Perspect. 1985, 60, 47-56.

Trosko, J .E. Michigan State University, East Lansing, MI. Personal communication,
2007.

Rummel, A.M.; Trosko, J .E.; Wilson, M.R.; Upham, B.L. Polycyclic Aromatic

Hydrocarbons with Bay-like Regions Inhibited Gap J unctional Intercellular
Communication and Stimulated MAPK Activity. Toxicol. Sci. 1999, 49(2), 232-240.

60

Chapter 3

Toxicity of Parent Polychlorinated Biphenyl (4,4'-Dichlorobiphenyl)

3.1 Introduction

Although polychlorinated biphenyl (PCB) contamination is typically encountered
as site-specific mixtures of PCB congeners, toxicity tests on individual PCB congeners
are important to conduct so that the toxicity of a particular mixture can be extrapolated,
based on the toxicity of its component congeners (1 ). In view of the cytotoxicity and
GJIC inhibitory effects observed for 4,4'-dichloro-3-biphenylol (4,4'DC3BP), the PCB
congener 4,4'-dichlorobiphenyl (4,4'DCBP) was selected for Fenton’s reagent
remediation studies. The biological significance of 4,4'DCBP is further indicated by the
results of metabolism studies in various organisms. In a study performed by Tulp et al.
(2), 4,4’DC3BP was found to be excreted as a major metabolite following the metabolism
of 4,4'DCBP by rats, rabbits, goats, frogs, and fungi. Further studies on the metabolism
of 4,4'DCBP in frogs revealed that both 4,4'DC3BP and 4,4'DCBP became “trapped” in
tissues (2). In studies of 4,4'DCBP in goats, the only urinary metabolite was identified as
4,4'DC3BP; however, this metabolite was absent in the feces and several organs (3). In
rats, the metabolites of 4,4'DCBP excreted in bile were identified as 3,4'-dichlorO—4-
biphenylol and 4,4'DC3BP (4). In studies of white-rot fungi Phanerochaete
chrysosporium and Phanerochaete sp. MZ142, 4,4’DCBP was metabolized to various
hydroxylated PCBs as a key step in the PCB degradation process (5).

Experimental results show that substitution in the ortho position from the carbon

bridge of the biphenyl is essential and at least one chloro substituent in the ortho position

61

is necessary for the ability to inhibit gap junctional intercellular communication (GJIC)
(6). PCBs lacking ortho-substituted chlorine atoms may have a ceplanar conformation,
whereas an increased number of chloro substituents in ortho positions has been shown to
result in decreased coplanarity (6). Although PCBs with coplanar conformations appear
less likely to inhibit GJIC, decreased coplanarity of the PCB increases the chances of
inhibition of GJIC (6-8). Since 4,4'DCBP lacks ortho-substituted chlorine atoms, a
coplanar conformation can be expected.

In this section of research, the toxicity of the parent PCB (4,4'DCBP) for the
F enton’s remediation studies was evaluated in rat liver epithelial cells using a
nongenotoxic bioassay that determines the in vitro modulation of GJIC as a measure of

the epigenetic toxicity.

3.2 Experimental Section
3.2.1 Chemicals

4,4'-dichlorobiphenyl (99.4% purity) was purchased from Chem Service Inc.
(West Chester, PA). Acetonitrile (ACN) (99.8% purity) was purchased from EM Science
(Gibbstown, NJ). Dimethyl sulfoxide (DMSO) (99.7% purity) and fatty acid free bovine
serum albumin (BSA) (96% purity) were purchased from Sigma Chemical Co. (St. Louis,
MO). Acetone (99.8% purity) was purchased from J .T. Baker (Phillipsburg, NJ). For
cell culture, D-medium (Formula No. 78-5470 EG), Fetal Bovine Serum (F BS), and
Gentamicin were purchased fi'om GIBCO Laboratories (Grand Island, NY). Lucifer
Yellow CH, dilithium salt, was purchased from Molecular Probes Inc. (Eugene , OR),

ICN Biomedicals Inc. (Aurora, OH), and Sigma Chemical CO. (St. Louis, MO).

62

Formaldehyde solution (3 7%) for the GJIC bioassays was purchased from J .T. Baker

(Phillipsburg, NJ).

3.2.2 Methods
3.2.2.1 Cell Culture Techniques

The cell culture techniques performed were identical to those described in
Chapter 2.2.2.2 for epigenetic toxicity studies of hydroxylated biphenyls and

hydroxylated polychlorinated biphenyls as potential remediation byproducts.

3.2.2.2 Solvent Evaluation for GJIC Bioassay

In the Chapter 2 toxicology studies, solutions of the six potential remediation
byproducts were prepared with 100% acetonitrile (ACN) as the solvent. Although
4,4'DCBP completely dissolved in the ACN solvent, when the solution was added to the
media in the cell culture dishes, an oily fihn and/or cloudiness appeared. Therefore,
ACN proved to be an inappropriate solvent for 4,4'DCBP due to the insolubility of the
4,4'DCBP upon application of the solution to the media in the cell culture dishes, thereby
hindering exposure of the cells to the toxicant. In experiments evaluating the effects of
various PCBs on GJIC, Hemming et al. (6) used dirnethyl sulfoxide (DMSO) or DMSO-
acetone (1 :1 by volume) as solvents for the preparation of test solutions. To determine
the best solvent for 4,4’DCBP for the toxicology studies, four solvents were tested: 100%
ACN, DMSO-ACN (1:1 by volume), DMSO, and DMSO-acetone (1:1 by volume). A 20
mM test solution of 4,4’DCBP was prepared in each solvent and mixed thoroughly (by

sonication). Test volumes of 15 or 30 uL of 4,4'DCBP test solution (corresponding with

63

150 uM and 300 uM doses, respectively) were then applied to 2 mL of D-medium
supplemented with 5% FBS in 35 mm diameter culture dishes lacking cell cultures. Each
solvent was evaluated based on the ability to produce a test solution in which 4,4'DCBP

dissolves completely and remains in solution when added to the media in the culture

dishes.

3.2.2.3 PCB “Transport” Using DMSO/BSA/PBS Solvent for GJIC Bioassay

One possible option to solving the insolubility problem of 4,4'DCBP for the
toxicology studies was to find a means of “transporting” the PCB to the cells attached to
the bottom of the culture dish. Most proteins are comprised of hydrophilic regions and a
hydrophobic domain to transport hydrophobic compounds to. specific biological sites (9).
In the body, hydrophobic foreign compounds, such as PCBs, are often transported in the
blood and to the tissues by binding with plasma proteins such as albumin (10, 11).
Likewise, serum albumin would be useful for “transporting” 4,4'DCBP to the cells in the
bioassays. Since serum albumin carries large quantities of fatty acid (12), which would
cause inhibition of GJIC ([3), fatty acid fi'ee bovine serum albumin (BSA) was selected
for use in 4,4'DCBP toxicology studies. Two solutions were combined to produce the
final 4,4'DCBP test solution: (1) 1 mL of 20 mM 4,4'DCBP in DMSO and (2) 9 mL of
2% BSA in phosphate buffered saline (PBS) (w/v). A 20 mM solution of 4,4'DCBP was
prepared in DMSO and mixed thoroughly (by sonication). In a 25 mL Erlenmeyer flask,
the 20 mM 4,4'DCBP/DMSO solution was slowly added to the 2% BSA/PBS solution.
Using a stir bar, the test solution was mixed thoroughly throughout the 4,4'DCBP/DMSO

solution addition to create a solution of white milky appearance (indicative of the

64

microparticles responsible for “transporting” the 4,4'DCBP to the cells). The final test

solution concentration of 4,4'DCBP in DMSO/BSA/PBS was 2 mM.

3.2.2.4 Vehicle Tolerance Test for DMSO/BSAIPBS

The vehicle tolerance test (dose-response) was performed to determine the largest
volume of the solvent DMSO/BSA/PBS that could be applied to cell cultures without
causing inhibition of gap junctional intercellular communication (GJIC). For the
DMSO/BSA/PBS vehicle tolerance test, two solutions were combined to produce the
final vehicle test solution: (1) 1 mL of DMSO and (2) 9 mL of 2% BSA in PBS (w/v). In
a 25 mL Erlenmeyer flask containing a stir bar, DMSO was slowly added to the 2%
BSA/PBS solution and mixed thoroughly to produce the vehicle test solution. Bioassays
of GJIC were performed on confluent cell cultures (usually 2 days of growth) grown in
35 mm diameter culture dishes containing 2 mL of D-medium supplemented with 5%
FBS (as described in Chapter 2.2.2.2). The scrape-loading/dye transfer (SL/DT)
procedure for determining the GJIC was adapted from the method described by El-Fouly
et al. (14) and is described in detail by Hemer et al. (15). Treatments with the
DMSO/BSA/PBS vehicle test solution and controls (no dose) were performed in
triplicates. Doses were applied directly to the dishes of confluent cell cultures and
allowed to incubate for 30 minutes (at 37 °C in a water-j acketed IR Autoflow Automatic
CO2 incubator (NUAIRE, Inc., Plymouth, MN) in a humidified atmosphere with 5% CO2
and 95% air) before assaying for GJIC. The vehicle test solution was evaluated over a

volume range of 0 to 350 uL.

65

All culture dishes were examined within 24 hours of the experiment completion.
Each culture dish of cells was digitally photographed such that the observed scrape
spanned the full horizontal width of the picture. A COHU High Performance Color CCD
Camera (Cohu, Inc., San Diego, CA) with a magnification of 200x under a Nikon
Diaphot-TMD epifluorescence phase-contrast microscope (Nikon Corp., Japan)
illuminated with a Nikon HB-10101AF Super High Pressure Mercury 100W lamp (Nikon
Corp., Japan), or a Nikon TE300 Eclipse Inverted Microscope (Nikon Corp., Japan) with
a Nikon HB-10103AF Super High Pressure Mercury 100W lamp (Nikon Corp., Japan)
was used.

The fluorescence of the Lucifer yellow dye was used to determine the distance the
dye traveled perpendicular to the scrape. This distance of dye travel was indicative of the
level of GJIC within the culture. Quantitative analysis of the distance of dye spread was
performed using NucleoTech GelExpert software (N ucleoTech Corp., Hayward, CA).
The distance of dye spread was measured in terms of the area of dye spread, by tracing
manually via free object quantification the area of farthest visible fluorescence. Since the
width of the photographed section was the same for every culture dish, measuring the
area of the dye spread was equivalent to measuring the distance of dye spread
perpendicular to the scrape. The area of dye spread for each vehicle test solution
treatment dish was compared to a group of cells that were exposed to no dose (controls)
under the same assay as the treated cells. For each dish treated with the vehicle test
solution, the fraction of the control was calculated as the area of dye spread in the treated
dish divided by the average area of dye spread in the triplicate set of control (no dose)

dishes. The results for each set of vehicle test solution treated triplicates were reported as

66

an average fi'action of the control (F 0C) 3: standard deviation (SD) determined at the
95% confidence interval (CI).

The level of GJIC in cells exposed to the vehicle test solution was assessed by the
decrease in communication of the cells as compared to the control group, which was
exposed to no dose of chemical. A decrease in FOC corresponds directly to a decrease in
GJIC (where the doses are not cytotoxic). Interpretations of GJIC results are consistent
with those of Luster-Teasley et al. (16) and Hemer et al. (15). Complete communication
(100%) between the cells is identified as a F OC value of 1.0 as seen in the control. A
FOC value greater than 0.9 is difficult to statistically distinguish from the control. FOC
values between 0.9 and 0.5 indicate partial inhibition of GJIC. A F 0C value less than or
equal to 0.5 is indicative of a significant amount of inhibition of GJIC, since this would
be representative of communication levels that are 50% or less than the normal
communication levels. F OC values between 0.3 and 0.0 are representative of complete
inhibition of GJIC. A F 0C value of 0.3 is usually used to represent complete inhibition,
as it corresponds to the width of a single row of cells with no dye spreading beyond its
boundaries (15). Controls, which received no dose of vehicle test solution, were also
performed as a means of evaluating a normal level of GJIC and the overall “health” of the
cells. Statistical analyses were performed by means of the t-test and One Way Analysis

of Variance (AN OVA) to compare the vehicle test solution results and control results.

3.2.2.5 Dose-Response Bioassay

Bioassays of GJIC were performed on confluent cell cultures (usually 2 days of

growth) grown in 35 mm diameter culture dishes containing 2 mL of D-medium

67

supplemented with 5% FBS (as described in Chapter 2.2.2.2). The scrape-loading/dye
transfer (SL/DT) procedure for determining the GJIC was adapted from the method
described by El-Fouly et al. (14) and is described in detail by Hemer et al. (15). A
detailed description of the spread of Lucifer yellow dye from the scrape to neighboring
cells can be found in Wilson et al. (I 7).

For reasons to be discussed later in this chapter, which determined that finther
toxicology studies were unnecessary, only dose-response GJIC bioassays were
performed. Test solutions of 2 mM 4,4’DCBP in DMSO/BSA/PBS
(4,4'DCBP/DMSO/BSA/PBS) were prepared as described in Section 3.2.2.3. The
vehicle control solution (DMSO/BSA/PBS) was prepared as described in Section 3.2.2.4.
Chemical treatments (4,4'DCBP/DMSO/BSA/PBS), controls (no dose), and vehicle
controls (DMSO/BSA/PBS) were performed in triplicates. Doses were applied directly
to the dishes of confluent cell cultures and allowed to incubate for 30 minutes, 2 hours, 6
hours, and 24 hours (at 37 °C in a water-jacketed IR Autoflow Automatic CO2 incubator
(NUAIRE, Inc., Plymouth, MN) in a humidified atmosphere with 5% CO2 and 95% air)
before assaying for GJIC. Vehicle controls were dosed with a volume of
DMSO/BSA/PBS corresponding to the largest volume of chemical dose tested in the
treatment dishes (300 uL). Since the volume change caused by the addition of the
chemical dose in each culture dish was significant (greater than 2%), the observed or
“true” dose was calculated by adjusting for the added dose volume. A “true” dose range
of 0 to 260.87 uM of 4,4'DCBP was tested and is reflected in the figures.

All culture dishes were examined within 24 hours of the experiment completion.

Each culture dish of cells was digitally photographed and analyzed by the methods

68

described previously in Section 3.2.2.4. The area of dye spread for each chemical
treatment dish was compared to a control group of cells that were exposed to
DMSO/BSA/PBS only (vehicle controls) under the same assay as the treated cells. For
each chemically treated dish, the fraction of the control was calculated as the area of dye
spread in the treated dish divided by the average area of dye spread in the triplicate set of
vehicle control dishes. The results for each set of chemically treated triplicates were
reported as an average fraction of the control (FOC) i standard deviation (SD)
determined at the 95% confidence interval (CI).

The level of GJIC in cells exposed to the chemical was assessed by the decrease
in communication of the cells as compared to the vehicle control groups, exposed to
DMSO/BSA/PBS only. A decrease in F OC corresponds directly to a decrease in GJIC
(where the doses are not cytotoxic). Interpretations of GJIC results are consistent with
Luster-Teasley et a1. (16) and Hemer et al. (15). Complete communication (100%)
between the cells is identified as a FOC value of 1.0 as seen in the vehicle control. A
FOC value greater than 0.9 is difficult to statistically distinguish from the vehicle
controls. F OC values between 0.9 and 0.5 indicate partial inhibition of GJIC. A F OC
value less than or equal to 0.5 is indicative of a significant amount of inhibition of GJIC,
since this would be representative of communication levels that are 50% or less than the
normal communication levels. F OC values between 0.3 and 0.0 are representative of
complete inhibition of GJIC. A F OC value of 0.3 is usually used to represent complete
inhibition, as it corresponds to the width of a single row of cells with no dye spreading
beyond its boundaries ([5). Controls, which received no close of chemical or solvent

(DMSO/BSA/PBS), were performed for each experiment as a means of evaluating a

69

normal level of GJIC and the overall “health” of the cells. By performing a t-test for each
experiment with incubation times of 30 minutes, 2 hours, 6 hours, and 24 hours, it was
found that the areas of dye spread for the control dishes (no solvent or chemical) did not
vary significantly from the areas for the vehicle controls (only DMSO/BSA/PBS) at a
95% CI. Therefore, it could be concluded that the solvent (DMSO/BSA/PBS), at the
volume tested and time of incubation, was not a significant source of inhibition in the
experiments. Statistical analyses were performed by means of the t-test and One Way
Analysis of Variance (ANOVA) to compare the chemical treatment results and vehicle

control results.

3.3 Results and Discussion
3.3.1 Solvent Evaluation for GJIC Bioassay

4,4'DCBP was determined to be completely soluble in all four solvents tested
(100% ACN, DMSO-ACN (1 :1 by volume), DMSO, and DMSO-acetone (l :1 by
volume) in preparation of the 20 mM test solutions. The test solutions were clear and
colorless with no 0in film or cloudiness. However, when 15 or 30 uL of the 20 mM
4,4'DCBP test solutions (corresponding with 150 uM and 300 uM doses, respectively)
were added to the culture dishes containing 2 mL of D-medium supplemented with 5%
FBS, an oily film on the surface of the media and/or precipitate/cloudiness was observed.
If these culture dishes had contained cell cultures, chemical contact with the cells would
have been hindered, since the cells would have been attached to the bottom surface of the
dish. Therefore, none of the solvents tested were appropriate for the toxicology studies

with 4,4'DCBP.

70

3.3.2 Vehicle Tolerance Test for DMSO/BSA/PBS

The DMSO/BSA/PBS vehicle test solution was clear and colorless with no oily
film, cloudiness, or milky appearance. When doses were added to the dishes of confluent
cell cultures, the media remained clear and colorless. As shown in Figure 3.1, the vehicle

tolerance test GJIC bioassay results with 30 minutes of incubation indicated no inhibition
of GJIC for a volume range of 0 to 350 uL of DMSO/BSA/PBS. It can be assumed,
therefore, that any inhibition of GJIC observed for doses of 4,4'DCBP containing no
more than 350 pL of the vehicle (DMSO/BSA/PBS) is not attributable to the vehicle, but
rather an effect of the presence of 4,4'DCBP. Volumes of DMSO/BSA/PBS greater than
350 uL were not investigated, since the maximum test volume of 2 mM

4,4'DCBP/DMSO/BSA/PBS was 300 11L.

3.3.3 Dose-Response Bioassay

The test solution of 2 mM 4,4'DCBP in DMSO/BSA/PBS had a white milky
appearance, while the vehicle control solution (DMSO/BSA/PBS) was clear and colorless
with no oily film, cloudiness, or milky appearance. When the vehicle control solution
was added to the dishes of confluent cell cultures, the media remained clear and colorless.
Although the media in the dishes of confluent cell cultures remained clear and colorless

for 4,4'DCBP at doses up to 76.92 M, at higher doses the media became more milky in

appearance.

As shown in Figures 3.2, 3.3, and 3.4, no significant level of inhibition of GJIC
was observed for 4,4'DCBP for a dose range of 0 to 260.87 uM for incubation times of

30 minutes, 2 hours, and 6 hours, respectively. The maximum levels of inhibition of

71

 

 

0.75 - -

GJIC
(Fraction of the Control)

0.50 - ..

0.25 "" d

P

l l l I L l
0 50 100 150 200 250 300 350

Volume (pL)

 

 

 

0.00

Figure 3.1 Vehicle tolerance test GJIC bioassay results with a 30 minute incubation time
for a volume range of 0 to 350 p.L of DMSO/BSA/PBS. Each data point is representative
of the results for a set of DMSO/BSA/PBS treated triplicates reported as an average FOC
i standard deviation determined at the 95% confidence interval.

72

 

 

 

 

l I l l l l
1.00 K. . . I . ___.-
E
E
o 0.75 -
2%
3:5 _
5 0.50
‘5
2
E:
0.25 -
000 L 1 L 1 I
0 50 100 150 200 250

00890”)

Figure 3.2 Dose-response bioassay results for 4,4'-dichlorobiphenyl (DMSO/BSA/PBS
solvent) with 30 minutes of incubation time for a dose range of 0 to 260.87 uM. Each
data point is representative of the results for a set of chemically treated triplicates
reported as an average F 0C i standard deviation determined at the 95% confidence
interval.

73

 

0.75 - .I

GJIC
(Fraction of the Control)

9
8
I

r

 

 

 

0.25 - -
0.00 l J l I l
0 50 100 150 200 250
Dose (uM)

Figure 3.3 Dose-response bioassay results for 4,4’-dichlorobiphenyl (DMSO/BSA/PBS
solvent) with 2 hours of incubation time for a dose range of 0 to 260.87 M. Each data
point is representative of the results for a set of chemically treated triplicates reported as
an average FOC 4: standard deviation determined at the 95% confidence interval.

74

 

 

 

 

I I I I I I
1.00 H\ | . 1
E
'5'
o 0.75 - -
2%
8‘5
.5 0.50 *- -
‘6
E
E:
0.25 - -
0.00 I I I I I I
0 50 100 150 200 250
Dose(pM)

Figure 3.4 Dose-response bioassay results for 4,4'-dichlorobiphenyl (DMSO/BSA/PBS
solvent) with 6 hours of incubation time for a dose range of O to 260.87 ”M. Each data
point is representative of the results for a set of chemically treated triplicates reported as
an average F 0C i standard deviation determined at the 95% confidence interval.

75

GJIC observed for 30 minutes, 2 hours, and 6 hours of incubation were F OC values of
0.94 i 0.01 at 39.22 uM, 0.99 i 0.02 at 139.53 uM, and 0.95 i 0.02 at 260.87 uM,
respectively. Figure 3.5 presents the dose-response GJIC bioassay results for 4,4'DCBP
for a dose range of 0 to 260.87 uM with an incubation time of 24 hours. Very slight
inhibition of GJIC occurred between doses of 0 to 95.24 uM of 4,4'DCBP with 24 hours
of incubation. The maximum level of inhibition of GJIC for 24 hours of incubation was
exhibited at a dose of 19.80 uM (FOC = 0.86 _+. 0.02). No significant level of inhibition
of GJIC was observed over a dose range of 139.53 to 260.87 “M for 24 hours of

incubation.

3.4 Conclusions

The dose-response results for 4,4'DCBP are consistent with observations that
PCBs with coplanar conformations appear less likely to inhibit GJIC (6-8). Although
4,4'DCBP was not observed to be significantly inhibitory to GJIC, Fenton’s remediation
of this chemical has the potential of generating byproducts which are inhibitory to GJIC
and/or cytotoxic (based on the potential of forming byproducts such as 4,4'DC3BP).
Likewise, Fenton’s remediation of a mixture of PCBs which included 4,4'DCBP might be
a matter of concern. Since very slight to no inhibition of GJIC was observed for
4,4'DCBP for the dose-response experiments for incubation times of 30 minutes, 2 hours,

6 hours, and 24 hours, no further toxicity studies were performed.

76

 

 

 

 

I I I I I I
1.00 -
"<5
'3'
o 0.75 — -'
2%
8'3
.5 0.50 - d
*6
8
it
0.25 - 4
0.00 I I I I I I
0 50 100 150 200 250
Dose(pM)

Figure 3.5 Dose-response bioassay results for 4,4’-dichlorobiphenyl (DMSO/BSA/PBS
solvent) with 24 hours of incubation time for a dose range of 0 to 260.87 uM. Each data
point is representative of the results for a set of chemically treated triplicates reported as
an average FOC i standard deviation determined at the 95% confidence interval.

77

3.5 References

1. National Research Council. A Risk-Management Strategy for PCB-Contaminated
Sediments. National Academy Press: Washington, DC, 2001.

2. Tulp, M.T.M.; Sundstrom, G.; Hutzinger, O. The Metabolism of 4,4’-
Dichlorobiphenyl in Rats and Frogs. Chemosphere. 1976, 5 (6), 425—432.

3. Safe, 8.; Platonow, N.; Hutzinger, O. Metabolism of Chlorobiphenyls in the Goat and
Cow. J. Agric. Food Chem. 1975, 23 (2), 259-261.

4. Hass, J .R.; J ao, L.T.; Wilson, N.K.; Matthews, H.B. Metabolism of 4-Chlorobiphenyl
and 4,4’-Dichlorobiphenyl in the Rat: Qualitative and Quantitative Aspects. .1 Agric.
Food Chem. 1977, 25(6), 1330-1333.

5. Kamei, 1.; Kogura, R.; Kondo, R. Metabolism of 4,4'-dichlorobiphenyl by white-rot
fungi Phanerochaete chrysosporium and Phanerochaete sp. MZ142. Appl. Microbiol.
Biotechnol. 2006, 72(3), 566-575.

6. Hemming, H.; Wamgard, L.; Ahlborg, U.G. Inhibition of Dye Transfer in Rat Liver
WB Cell Culture by Polychlorinated Biphenyls. Pharmacol. Toxicol. 1991, 69(6),
416-420.

7. Silberhom, E.M.; Glauert, H.P.; Robertson, L.W. Carcinogenicity of Polyhalogenated
Biphenyls: PCBs and PBBs. Crit. Rev. T oxicol. 1990, 20(6), 440-496.

8. Kang, K.S.; Wilson, M.R.; Hayashi, T.; Chang, C.C.; Trosko, J .E. Inhibition of Gap
Junctional Intercellular Communication in Normal Human Breast Epithelial Cells
afier Treatment with Pesticides, PCBs, and PBBs, Alone or in Mixtures. Environ.
Health Perspect. 1996, 104 (2), 192-200.

9. Branden, C.; Tooze, J. Introduction to Protein Structure, Second Ed. Garland
Publishing, Inc.: New York, NY, 1999,14.

10. Timbrell, J. A. Principles of Biochemical Toxicology, Second Ed. Taylor & Francis
Inc.: Bristol, PA, 1991, 49-51.

11. Muhlebach, S.; Wyss, P.A.; Bickel, M.H. The use of 2,4,5,2',4',5'-
Hexachlorobiphenyl (6-CB) as an Unmetabolizable Lipophilic Model Compound.
Pharmacol. Toxicol. 1991, 69(6), 410-415.

12. Voet, D.; Voet, J .G. Biochemistry, Second Ed. , John Wiley & Sons, Inc.: New York,
NY, 1995, 666.

78

13.

14.

15.

16.

17.

Hayashi, T.; Matesic, D.F.; Nomata, K.; Kang, K.-S.; Chang, C.C.; Trosko, J .E.
Stimuation of cell proliferation and inhibition of gap junctional intercellular
communication by linoleic acid. Cancer Lett. 1997, I 12, 103-111.

El-Fouly, M.H.; Trosko, J .E.; Chang, C.C. Scrape-Loading and Dye Transfer: A rapid
and simple technique to study gap junctional intercellular communication. Exp. Cell
Res. 1987, 168, 422-430.

Hemer, H.A.; Trosko, J .E.; Masten, S.J. The Epigenetic Toxicity of Pyrene and
Related Ozonation Byproducts Containing an Aldehyde Functional Group. Environ.
Sci. T echnol. 2001, 35 (17), 3576-3583.

Luster-Teasley, S.L.; Yao, J .J .; Hemer, H.A.; Trosko, J .13.; Masten, S.J. Ozonation of
Chrysene: Evaluation of Byproduct Mixtures and Identification of Toxic Constituent.
Environ. Sci. T echnol. 2002, 36(5), 869-876.

Wilson, M.R.; Close, T.W.; Trosko, J .E. Cell Population Dynamics (Apoptosis,
Mitosis, and Cell-Cell Communication) during Disruption of Homeostasis. Exp. Cell
Res. 2000, 254, 257-268.

79

Chapter 4

Methylene Blue Dye Test for Rapid Qualitative Detection of Hydroxyl Radicals
Formed in a Fenton’s Reaction Aqueous Solution

4.1 Introduction

An article (I) based on this chapter was published in Environmental Science and
Technology and a US. Patent Application, Serial No. 11/478,959, has been filed.

Several methods have been developed to detect hydroxyl radicals; however, none
can rapidly and qualitatively determine hydroxyl radicals by a simple procedure requiring
inexpensive materials and without interferences with the reaction. Previous methods
developed for the quantitation/detection of hydroxyl radicals often depend on the addition
of chemical probes, such as benzoic acid, l-propanol, or salicylic acid, and the
hydroxylated reaction products have been used as an indirect measurement of the
presence of hydroxyl radicals (2-4). These methods involve complicated, time-
consurning procedures, are non-specific for the hydroxyl radical, and were often
developed for a specific experimental study or system. Furthermore, in the presence of
other hydroxyl radical scavenging/reacting chemicals, the addition of a chemical probe
results in competition for the hydroxyl radicals and the possibility of side reactions. The
numerous hydroxylated products formed often make the quantitative detection of
hydroxyl radicals complicated. Another commonly used method, electron spin resonance
(ESR), capable of detecting hydroxyl radicals and superoxide anion radicals, requires
expensive instrumentation, is laborious, and may involve a number of transient radicals
(5). Although in iron/hydrogen peroxide (F enton’s reagent) systems, some researchers

have measured the loss of hydrogen peroxide as an indicator for hydroxyl radical

80

formation, only a fraction of the peroxide degraded is converted to hydroxyl radicals (as
the complexity of the reaction leads to the formation of a variety of species) (2).

The methylene blue (MB) dye test is a new test that qualitatively indicates the
presence of hydroxyl radicals through an immediate, distinct bleaching of the MB dye on
a paper test strip afier applying an aqueous sample containing hydroxyl radicals. This
method is simple, requires inexpensive materials, and does not suffer fiom interferences
resulting from the addition of probe chemicals. Methylene blue
(3,7-bis(dimethylamino)phenothiazin-5-ium chloride) is a basic dye of the thiazine series
used extensively for dyeing and printing cloth and for medicinal purposes (based on its

antiseptic properties) (6). The structure of methylene blue is shown in Figure 4.1 (7).

_ N
C/ 0
Cl -
\
(CH3)2N s N(CH
_ +

3)2

—

 

 

Figure 4.1 Structure of Methylene Blue

In the MB dye test, the hydroxyl radical reacts with the MB cation to produce a
hydroxide ion and a MB radical cation (8). Since the MB cation is dark blue in color and
the MB radical cation is colorless, application of a sample containing hydroxyl radicals to
MB dye results in a change of color from dark blue to colorless. Hydrogen peroxide
(3%) does not cause bleaching of MB dye.

A Fenton system was used as the source of hydroxyl radicals to test the
applicability of the MB dye test for the detection of hydroxyl radicals. Highly reactive

hydroxyl radicals are produced by the oxidation of ferrous iron and the reduction of

81

hydrogen peroxide (9). Hydrogen peroxide is added in the presence of ferrous iron to a
solution or suspension of compounds to be treated (10). The oxidation efficiency of the
Fenton’s type reactions depends on the Fe2+zH202 ratio and the pH (11). The optimal pH
for the Fenton’s reaction efficiency has been shown to be between pH 3 and 5 (12). At
more basic pH values, the iron is converted from a hydrated ferrous form to a colloidal
ferric form, thereby causing a decrease in the effectiveness of the reaction (12). Fenton’s
reagent is an effective method of remediating contaminated soils and aqueous solutions
through the oxidation by hydroxyl radicals, which readily degrade a wide variety of
organic pollutants (9). Rapid, inexpensive detection of the presence of hydroxyl radicals
would allow for the immediate monitoring of the Fenton’s remediation process and aid in
optimizing the degradation efficiency.

The objective of this study was to test the applicability of the MB dye test for the
detection of hydroxyl radicals in a Fenton’s reaction aqueous solution and verify the
results by benzoic acid chemical probe hydroxyl radical detection methods using thin

layer chromatography and spectrophotometric wavelength scans.

4.2 Experimental Section
4.2.1 Chemicals

Methylene blue dye (3,7-bis(dimethylamino)phenothiazin—5 -ium chloride) (97%
purity) was purchased from F luka (Buchs, Switzerland). Thirty-percent hydrogen
peroxide (HzOz) (unstabilized), iron (11) sulfate heptahydrate (F eSO4-7H20 ) (99%
purity), sodium sulfite (N a2SO3) (anhydrous, 98% purity), benzoic acid (BA) (99.5%

purity), and 4-hydroxybenzoic acid (4—HBA) (99+ % purity) were purchased from

82

Sigma—Aldrich (St. Louis, MO). Sulfuric acid (H2804) (96% purity), sodium hydroxide
(NaOH) pellets (99% purity), methanol (MeOH) (100%), and chloroform (100%) were
purchased from J .T. Baker (Phillipsburg, NJ). Throughout this chapter and dissertation,
Milli-Q water was obtained from a Milli-Q Ultrapure Water Purification System (System

Type ZMQS6VFOY) purchased from Millipore Corp. (Bedford, MA).

4.2.2 Methods
4.2.2.1 Methylene Blue Dye Test

Prior to the development of the methylene blue dye test strip, a liquid methylene
blue dye test was investigated. In the liquid methylene blue dye test, 60 uL of a liquid
test sample was added to a 0.2 mL PCR vial containing 100 uL of 0.1 mM methylene
blue dye solution. The solution was then completely mixed by inverting the closed vial.
This liquid methylene blue dye test was later abandoned; however, since the color change
of the liquid methylene blue dye due to the presence of hydroxyl radicals occurred only
after a lengthy period of time, approximately 40 minutes. These results are consistent
with those observed by Dutta et al. (6), where oxidation of methylene blue in aqueous
solution by hydroxyl radicals required 1 hour to achieve 98% discoloration of the dye.
The methylene blue dye test strip was developed as an alternative to this liquid method
and allowed for the immediate detection of hydroxyl radicals. In a liquid phase reaction,
such as the liquid methylene blue dye test, where molecules are allowed to collide in 3-
dimensional space, the reaction is dependent on the concentration of the colliding species.
In the dye test strip, since the methylene blue dye is soaked into and onto the filter paper

fibers, the oxidation of methylene blue by the hydroxyl radicals is aided by the greater

83

surface area available for the reaction and a higher concentration, resulting in a higher
rate of reaction.

To standardize the MB dye test, test strips were developed such that each had a
consistent, uniform MB dyed section on which samples could be applied. A 1.0 mM MB
dye solution was prepared in Milli-Q water from a 10 mM stock solution prepared with
methanol. Qualitative filter paper (Grade 1, 70 mm diameter circles, medium porosity)
(Fisher Scientific, Hanover Park, IL) was cut into two rectangular test strips
approximately 2 cm by 6 cm in size. Using a black, fine point, industrial strength,
permanent marker, a horizontal line was made on both sides of the test strip about 1.5 cm
fiom the bottom and allowed to dry. This marker line serves as a hydrophobic barrier
that prevents the MB dye from spreading above this line during the dipping process. The
bottom of the test strip was then dipped 10 times into 1.0 mM MB dye solution to the
level of the marker line. The dipped test strip was then placed onto a paper towel-lined
tray and allowed to completely dry in the dark. Dried strips stored for 24 hours were
used in this study; however, strips can be stored in a sealed, dark plastic bag for up to 33
days without adversely affecting test results.

The MB dye test was performed during the Fenton’s reaction to verify the
formation of hydroxyl radicals and dming the quenching process to verify completion of
quenching. Forty nricroliters of a liquid sample were placed dropwise onto the center of
the MB dyed section of a test strip, allowing for absorption between drops. All MB dye
tests were compared against a test strip tested with Milli-Q water. The absence of
bleaching of the MB dye indicated that no hydroxyl radicals were present to the extent

detectable by this qualitative test. In the quenching process of the Fenton’s reaction, the

84

absence of bleaching signified that quenching was complete. Bleaching of the MB dye,
due to the presence of hydroxyl radicals in a sample, was indicated by an immediate
discoloration of the MB dye fi'om a dark blue color to an almost white color, concentrated

at the point of application, with a dark blue outline.

4.2.2.2 Fenton’s Reaction in Milli-Q Water

A F enton’s reaction was performed to generate hydroxyl radicals in aqueous
samples. All experiments were performed in Milli-Q water. For the Fenton’s reaction, a
Fe2+zH202 molar ratio of 1:20 was used. The initial concentrations of Fe2+ and H202 in
the reaction mixture were 0.15 mM and 3 mM, respectively, which are within the ranges
previously used by Trapido et al. (10). For optimal Fenton’s reaction efficiency, the
F eSO4 solution was adjusted to pH 3 with 0.5 M H2804 and/or 1M NaOH. The pH was
monitored using a 720A plus pH/ISE meter with an 8102 BNU Ross Ultra Combination
pH electrode (ThermoOrion, Beverly, MA).

The F enton’s reaction was initiated by the addition of 3% H202, prepared from
30% unstabilized H202 and Milli-Q water, to the reaction mixture to obtain an initial
concentration of 3 mM H202. Since stabilizing agents (hydroxyl radical scavengers) in
commercial H202 might affect the results (13), only H202 devoid of stabilizing agents
was used. In order to prevent localized reactions (that might occur when a small volume
of very concentrated solution is added to a reaction mixture), 3% H202 rather than 30%
H202 was used to initiate the reaction. To test, qualitatively, the production of hydroxyl
radicals during the reaction, MB dye tests were performed on the unquenched reaction

mixture at 15, 30, and/or 60 minutes of reaction.

85

After 60 minutes, the Fenton’s reaction was quenched with a 10% Na2803
solution (w/v), prepared from Na2SO3 and Milli-Q water. Trapido et al. (10, 14)
recommended quenching by the addition of 2 to 3 drops of 10% Na2803 solution for
every 10 mL of reaction mixture. To verify, qualitatively, that quenching was complete,
5 minutes after the addition of the 10% Na2803 solution, a MB dye test was performed.
Additional 10% NaZSO3 solution was added to the reaction mixture and the MB dye test
was repeated until no discoloration of the MB dye was observed (quenching was

complete).

4.2.2.3 Hydroxyl Radical Detection by Benzoic Acid

To verify the ability of the MB dye test to detect the presence of hydroxyl radicals
in a Fenton’s reaction, experiments were performed using benzoic acid (BA) as a
chemical probe. BA reacts with hydroxyl radicals to form o-, m-, and p-hydroxybenzoic
acids as well as other products (2, 15). The presence of hydroxyl radicals can be
indirectly determined through the detection of these hydroxylated benzoic acids (HBAs)
using thin layer chromatography (TLC) and spectrophotometric wavelength scans.

Two Fenton’s reaction experiments were performed with the addition of BA.
Based on studies performed by Lindsey et al. (2), finely ground BA was added to the
Fenton’s reaction solution to obtain a final concentration of 9 mM BA. The experiment,
“Benzoic Acid in an Unquenched Fenton’s Reaction Mixture,” was similar to the
procedure of the “Fenton’s Reaction in Milli-Q Water” prior to quenching, except that
BA was added immediately preceding the initiation of the Fenton’s reaction. Following

initiation, the reaction was monitored using TLC and spectrophotometric Wavelength

86

scans at various times throughout the reaction period (120 minutes). The objective was

to verify the presence of hydroxyl radicals in the Fenton’s reaction mixture, as was
previously detected by the MB dye test. The second experiment, “Benzoic Acid in a
Quenched F enton’s Reaction Mixture,” was identical to the procedure of the “Fenton’s
Reaction in Milli-Q Water,” except that BA was added following the completion of
quenching. TLC and spectrophotometric wavelength scans were performed at various
times until 90 minutes had elapsed. A wavelength scan of the reaction mixture was also
performed immediately preceding the addition of BA. The objective was to verify the
absence of hydroxyl radicals in the F enton’s reaction mixture following the completion of

quenching, as was previously detected by the MB dye test.

4.2.2.4 Thin-Layer Chromatography (TLC)

TLC was performed by the procedure described in Skoog et al. (16). TLC plates
were spotted using Drummond 1 ILL “Microcap” micropipettes (Fisher Scientific,
Hanover Park, IL) onto 2.5 x 7.5 cm silica gel 60 F254 precoated TLC plates with acid-
stable fluorescent indicator (Fisher Scientific, Hanover Park, IL). The left side of the
TLC plate was spotted with one of three selected standard solutions: 2 uL of 9 mM
BA/MeOH, 1 ILL of 9 mM 4-HBA/MeOH, or 2 )JL of a mixed standard consisting of 50
uL 9 mM BA/MeOH and 25 uL 9 mM 4-HBA/MeOH. A volume of 3 uL of Fenton’s
reaction sample was spotted on the right side of the TLC plates. The origin, where the
standard and sample were spotted, was marked near the bottom of the plate by two
horizontal lines on either side of the plate. Although 12 different eluting solvents (Table

4.1) were investigated for developing the TLC plates, a solution of methanolzchloroform

87

at a volumetric ratio of 2: 10 was selected as the eluting solvent, since it resulted in the

best separation of BA and 4-HBA.

Table 4.1 Potential Eluting Solvents Investigated for TLC Plate Development

 

 

 

 

Eluting Solvent Volumetric Ratio
Butanol:WaterzAcetic Acid 60:25:15
Methanol :Water 50: 50
Ethanol:Water 50:50
Isopropanol:Water 50:50
Butanol:Water 5 0:50
Methanol :Chloroforrn 1 :10
Dichloromethane:Methanol 10: 1
Ethyl Acetate:n-Hexane 50:50
n-Hexane:Ethanol:Ethyl Acetate 80:10:10
Methanol:Chloroform 1 :12
Methanol:Chloroform l :40
Methanol:Chloroform 2:10

 

 

TLC plates were examined under ultraviolet light (254 nm) and the resulting
“spots” were carefully traced with a pencil with a dot marked in the center of maximum
intensity. Retention factors (Rf) for each separated compound were calculated as the
distance traveled by the compound measured to the point of maximum intensity divided
by the distance traveled by the solvent front. The Rf value of each compound separated
from the Fenton’s reaction sample was compared with the Rf value of the standard on the
same plate (I 7). An absolute Rf difference of greater than 0.05 was considered to be a
significant difference and indicated that the compound was different from the standard.

An additional experiment, “TLC of an Unquenched Fenton’s Reaction Mixture
without BA Addition,” was similar to the procedure of “Fenton’s Reaction in Milli-Q

Water” prior to quenching. Following initiation, the reaction was monitored by

88

performing TLC at various times. The objective was to verify that the TLC results of
“Benzoic Acid in an Unquenched Fenton’s Reaction Mixture” were due to the reaction of

BA with hydroxyl radicals.

4.2.2.5 Spectrophotometric Wavelength Scans
Fenton’s reaction samples were scanned spectrophotometrically in cuvettes (polystyrene,
optical pathlength of 10 mm) on a Beckman DU7400 spectrophotometer (Beckman

Instruments, Fullerton, CA). Milli-Q water was used as a blank.

4.3 Results and Discussion
4.3.1 Fenton’s Reaction in Milli-Q Water (N o Benzoic Acid Addition)

The MB dye test was performed to determine the presence of hydroxyl radicals
generated by the Fenton’s reaction. As shown in Figure 4.2A, the MB dye test control
strip (no sample added) was homogeneously dark blue in color. When the MB dye test
was performed with Milli-Q water (Figure 4.2B), no bleaching or discoloration was
observed. During the Fenton’s reaction experiment prior to quenching, no significant
change in solution pH (~ pH 3.0) and temperature (23.0 °C) occurred during the 60
minute reaction period, and the solution remained clear and colorless in appearance. As
shown in Figures 4.2C and 4.2D, strips tested with unquenched Fenton’s reaction mixture
at 15 and 30 minutes, respectively, indicated the presence of hydroxyl radicals by
immediate bleaching.

The Fenton’s reaction was quenched after 60 minutes. The pH of the reaction

mixture increased from 3.1 to 7.7. The temperature was 22.5 °C. The color of the

89

Figure 4.2 Methylene blue dye test results for Fenton’s reaction in Milli-Q water. For
test strips (A) control (no sample added) and (B) 40 uL of Milli-Q water, no bleaching or
discoloration of the methylene blue dye is observed. Test strips with 40 uL of
unquenched Fenton’s reaction mixture at (C) 15 minutes and (D) 30 minutes of reaction
indicate the presence of hydroxyl radicals by bleaching of the methylene blue dye from
dark blue to an almost white color with a dark blue outline. Test strips with 40 uL of
Fenton’s reaction mixture quenched with (E) 30 drops and (F) 35 drops of 10% NazSO3
indicate the incomplete quenching and absence of hydroxyl radicals by very slight
bleaching and no bleaching, respectively.

90

.,
A}
-~_. ‘. _/
‘- ._.
N .2 .“
r , “f
I.
".
E
I
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Hf /
._ II fir . ,
i t
." ’. 3. ' 'An. '_..
4m '7 ’
1 ‘ r
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r 91». ‘.‘ I W -.
' ) r 'v’r‘ '. 7'
l 1 t
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reaction mixture changed fiom colorless to light orange, which can be attributed to the
more basic pH of the reaction mixture during the quenching process, resulting in the
conversion of iron fi'om a hydrated ferrous form to a colloidal ferric form and the
formation of ferric hydroxide (2, 18, 19). As shown in Figure 4.2E, a MB dye test of the
F enton’s reaction mixture quenched with 30 drops (1.23 mL) of quenching agent (10%
NaZSOg) produced very slight bleaching as indicated by light blue discoloration with no
dark blue outline. As shown in Figure 4.2F, the addition of 5 more drops (0.21 mL) of
10% Na2803 to the reaction mixture resulted in a complete absence of bleaching,

indicating that quenching was complete and no hydroxyl radicals remained.

4.3.2 Benzoic Acid in an Unquenched Fenton’s Reaction Mixture

An experiment involving Fenton’s reaction and the addition of BA was performed
to verify the presence of hydroxyl radicals. Since BA is only slightly soluble in water at
pH 3, the remaining BA was allowed to remain floating on the liquid surface or
suspended in solution. The addition of BA to the reaction mixture did not significantly
alter the pH. Following initiation of the F enton’s reaction by addition of 3% H202, the
reaction mixture appearance changed from colorless to a light pink color. No significant
change in solution pH (~ pH 3.0) and temperature (23.0 °C) occurred during the 120
minute reaction period.

The reaction mixture continued to darken in color to violet (at 30 minutes) and
eventually dark violet (at 60 minutes), corresponding to the gradual dissolution of BA.
Complete dissolution of BA occurred after 90 minutes through the reaction with hydroxyl

radicals to form more soluble HBAs. One possible explanation for the change in reaction

92

mixture color to dark violet is the hydroxylation of BA by hydroxyl radicals to form
salicylic acid (2-hydroxybenzoic acid), followed by the formation of a
tetraaquosalicylatroiron (III) complex with Fe3+ (20). This violet complex is formed
under an acidic pH (21) and is characterized by peak absorption at a wavelength of 520
nm (22).

Following the addition of 3% H202 to the reaction mixture, the reaction was
monitored using TLC, as presented in Figure 4.3. The first two TLC plates, Figures 4.3A
and 4.3B, were spotted with the standards 9 mM BA/MeOH and 9 mM 4-HBA/MeOH,
respectively. Based on the results of these plates, the BA can be distinguished from the
4-HBA. The remainder of the TLC plates (Figures 4.3C-4.3F) was spotted with the
mixed standard. For the TLC plates in Figures 4.3A to 4.3C, the Fenton’s sample was
obtained afier 30 minutes of reaction, while for Figures 4.3D, 4.3E, and 4.3F, the
sampling times were 60, 90, and 120 minutes, respectively. The elution time for each
TLC plate was approximately 10 minutes.

The BA consistently traveled firrther on the plate than the 4-HBA, which was
darker in intensity than the BA. The products present in the reaction mixture changed as
the reaction proceeded. The TLC plates at 30 minutes and 60 minutes, Figures 4.3C and
4.3D, respectively, indicated one major product in a larger amount and numerous
products present in smaller amounts (a series of smaller connected spots). However, at
90 minutes, Figure 4.3B, one major product in a larger amount, numerous products in
smaller amounts, and two secondary products were observed. At 120 minutes, Figure
4.3F, two major products were observed with numerous products present in smaller

amounts.

93

Figure 4.3 Thin layer chromatography results from “Benzoic Acid in an Unquenched
Fenton’s Reaction Mixture.” The left side of each TLC plate was spotted with samples of
standards. Figures 4.3A and 4.3B, were spotted with the standards 9 mM benzoic acid
(BA) in methanol (MeOH) and 9 mM 4-hydroxybenzoic acid (HBA) in methanol,
respectively. The remainder of the TLC plates (Figures 4.3C-4.3F) was spotted with 2

uL of a mixed standard consisting of 50 uL 9 mM BA/MeOH and 25 uL 9 mM
HBA/MeOH. The right side of each TLC plate was spotted with a 3 uL sample of
Fenton’s reaction mixture fiom a particular reaction time. For Figures 4.3A to 4.3C, the
F enton’s sample was from 30 minutes of reaction, while for Figures 4.3D, 4.3B, and

4.3F, the sampling times were 60, 90, and 120 minutes, respectively.

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For all the F enton’s samples, none of the major spots correlated with either the
BA or 4-HBA standards, suggesting that the major spots are more likely HBA products
other than 4-HBA. Retention factor (Rf) values were only calculated for major spots and
are presented in Table 4.2 (Plates A-F). The spots were numbered with the spot of
farthest migration labeled as l and the remaining spots numbered consecutively. For all
the Fenton’s samples, the Rf values of the major spots were significantly different (>
0.05) from the Rf values calculated for BA and 4-HBA standards on the same plate.
However, the visual correspondence between the numerous products present in smaller
amounts and the 4-HBA standard spot suggests that 4-HBA was produced in a small
quantity. The presence of I-[BAs on the developed TLC plate for the F enton’s reaction
mixture sample verified that hydroxyl radicals were present in the reaction solution to
react with the added BA. Comparing TLC plates, based on the absolute difference in the
Rf values, spot 1 of the Fenton’s samples from 30, 60, 90, and 120 minutes of reaction
time are most likely the same compound. Furthermore, spot 2 of the Fenton’s sample
from 120 minutes is most likely the same compound as spot 3 of the Fenton’s sample
fi'om 90 minutes.

The change in color of the Fenton’s reaction mixture to dark violet was monitored
by spectrophotometric wavelength scans. Figure 4.4 presents the wavelength scans over
a range of 450 nm to 750 nm on 1000 uL samples of unquenched F enton’s reaction
mixture containing BA at 0 minutes to 120 minutes of elapsed reaction time. As the
reaction time increased, there was an increase in the absorbance value for each
wavelength correlating with the increase in the intensity of the violet color of the reaction

mixture. The maximum absorbance value (peak absorbance) occurred at a wavelength of

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Figure 4.4 Wavelength scans (absorption spectra) of unquenched Fenton’s reaction
mixture with a benzoic acid chemical probe. Wavelength scans were performed over a
wavelength range of 450 nm to 750 nm. Each wavelength scan represents a Fenton’s
reaction mixture sample at a different reaction time ranging from 0 minutes to 120
minutes after initiation of the Fenton’s reaction. Absorbance vs. reaction time of
unquenched F enton’s reaction mixture with a benzoic acid chemical probe at the
wavelength of 517 nm is shown in the inset. Each data point is representative of the
absorbance at 517 nm obtained from the wavelength scan of the Fenton’s reaction
mixture at a particular reaction time. Polystyrene cuvettes with an optical pathlength of
10 mm were used.

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517 nm as the tetraaquosalicylatroiron (III) complex was generated. The violet color of
the reaction mixture and the peak absorbance at a wavelength close to 520 nm are
indicative of the formation of HBA products and verify the presence of hydroxyl radicals.
The Figure 4.4 inset illustrates the increase in absorbance at 517 nm with an increase in
the reaction time.

After performing a wavelength scan on a 1000 uL sample of the unquenched
Fenton’s reaction mixture containing BA at 45 minutes reaction time, 25 ILL of 10%
Na2803 was mixed into the sample in the cuvette. The violet color of the solution
immediately changed to light yellow. A wavelength scan was then repeated over a range
of 400 nm to 750 nm. Figure 4.5 presents the wavelength scan of the unquenched
Fenton’s reaction mixture containing BA after 45 minutes of reaction time alongside the
wavelength scan of the same sample following quenching with 10% NaZSO3. Unlike the
unquenched sample scan for which a peak absorbance occurred at 517 nm, for the
quenched sample there was a loss of this peak corresponding with the disappearance of
the violet color. The disappearance of the violet color can be explained as the reduction
of Fe3+ to F e2+ by Na2803 (reducing agent), resulting in loss of the
tetraaquosalicylatroiron (III) complex. In addition, the NaZSO3 quenched the hydroxyl
radicals in the reaction mixture, thereby preventing further formation of salicylic acid by
the hydroxylation of BA. The light yellow color can be attributed to the more basic pH
of the reaction mixture during the quenching process as previously described. Since the
formation of the tetraaquosalicylatroiron (III) complex requires acidic conditions (21),
the presence of Fe“ in the quenched reaction mixture does not result in the reforming of

the complex.

100

Figure 4.5 Wavelength scans of the effect of quenching an unquenched Fenton’s reaction
mixture containing a benzoic acid chemical probe at 45 minutes of reaction time.
Wavelength scans were performed over a wavelength range of 400 nm to 750 nm. After
performing a wavelength scan on a 1000 uL sample of unquenched F enton’s reaction
mixture from 45 minutes of reaction time, 25 uL of 10% sodium sulfite quencher was
added to the sample. Following mixing, a wavelength scan was performed on the
quenched sample. Polystyrene cuvettes with an optical pathlength of 10 mm were used.

101

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4.3.3 TLC of an Unquenched Fenton’s Reaction Mixture without BA Addition

To verify that the unquenched TLC results of the preceding section were due to
the reaction of BA with hydroxyl radicals, the same experiment was repeated without the
addition of BA. No significant change in solution pH (~ pH 3.0) and temperature (22.5
°C) occurred throughout the 60 minute reaction period. The reaction mixture remained
clear and colorless following initiation of the F enton’s reaction, indicating that the
change in color to violet depends on the presence of both BA and hydroxyl radicals. This
observation was further supported by the “Benzoic Acid in an Unquenched F enton’s
Reaction Mixture” experiment, in which the pink color did not form in the presence of
BA until after the addition of 3% H202, resulting in the generation of hydroxyl radicals.
No spots appeared on the TLC plate for the F enton’s reaction mixture sample at 30 and
60 minutes of reaction time in the absence of BA, indicating that the spots observed
above the origin on the developed TLC plates for the “Benzoic Acid in an Unquenched
Fenton’s Reaction Mixture” experiment were a result of the reaction of BA with hydroxyl

radicals.

4.3.4 Benzoic Acid in a Quenched Fenton’s Reaction Mixture

An experiment, involving F enton’s reaction and the addition of BA following the
completion of quenching, was performed to verify the absence of hydroxyl radicals in the
reaction mixture. In the reaction prior to quenching, the pH (~ pH 3.0) and temperature
(22.0 °C) remained constant, and the reaction mixture remained clear and colorless.
After 60 minutes of reaction time, the Fenton’s reaction was quenched, and the reaction

mixture changed to light orange, attributable to the more basic pH of the reaction mixture

103

as a result of the quenching process. The pH ranged from 7.5 to 7.6 and the temperature
remained at 21.0 °C. MB dye tests were performed to determine the completion of
quenching, which occurred after the addition of 40 drops (1.76 mL) of 10% Na2803.

Following the addition of BA to the reaction mixture, the pH slowly decreased
from 7.6 to 4.6 after 90 minutes, and the temperature remained constant at 21.0 °C. The
BA dissolved in the reaction mixture slightly faster than what was observed in the
“Benzoic Acid in an Unquenched Fenton’s Reaction Mixture” experiment, achieving
complete dissolution in 60 minutes. The more rapid dissolution of BA can be explained
by the higher solubility of BA at a pH of 7.6 versus that at a pH of 3. The reaction
solution remained light orange following the addition of BA after quenching, firrther
supporting that the pink to violet color change is dependent on the presence of both BA
and hydroxyl radicals. In the absence of hydroxyl radicals and at a basic pH, salicylic
acid, and ultimately the tetraaquosalicylatroiron (III) complex, cannot form.

Following the addition of BA, the reaction was monitored using TLC. For all
Fenton’s reaction mixture samples, only one spot appeared on the TLC plate above the
origin and correlated with BA, as supported by the Rf calculations presented in Table 4.2
(Plates G-J). The F enton’s reaction mixture spot had an Rf range of 0.49 to 0.52. Since
the maximum absolute Rf difference between the Fenton’s reaction mixture spot and the
BA standard spot on the same plate was 0.04, these spots are assumed to be the same
compound. The absence of HBAs on the TLC plate results for the Fenton’s reaction
mixture sample verified that quenching was complete, and no hydroxyl radicals were

present in the solution when BA was added.

104

The absence of hydroxyl radicals following quenching was also assessed by
spectrophotometric wavelength scans over a wavelength range from 400 nm to 750 nm.
Prior to the BA addition but after completion of quenching, a wavelength scan was
performed on the reaction mixture to compare to the effect of BA. The results of this
wavelength scan, identified as “Pre-BA,” as well as scans performed on 1000 uL samples
of quenched Fenton’s reaction mixture from 0 to 90 minutes after the addition of BA, are
presented in Figure 4.6. The wavelength scan results, following the addition of BA, did
not vary significantly from that observed for “Pre-BA.” The absence of peak absorbance
at close to 520 nm was an indication of the lack of HBA products in the reaction mixture
and suggests the absence of hydroxyl radicals. Since no hydroxyl radicals were present
in the quenched reaction mixture to react with the added BA, the tetraaquosalicylatroiron

(III) complex did not form.

4.3.5 Methylene Blue Dye Test Strip Studies: Age, 3% H202, and Influence of pH

Three additional experiments were performed (Figure 4.7) to test the performance
of the methylene blue dye test strips. In the first experiment (Figure 4.7A), the effect of
age of the methylene blue dye test strip on test results was evaluated by applying 40 uL
of the same unquenched Fenton’s reaction solution (30 minutes of reaction time) to strips
of different ages (4 days and 33 days old). The unquenched Fenton’s reaction solution
was prepared as described previously in Section 4.2.2.2 (“F enton’s Reaction in Milli-Q
Water”). Since there was no significant difference between the degree of bleaching for
the 4 day and 33 day old test strips, age of the test strip does not appear to affect the

performance of the methylene blue dye test. It should also be noted that the degree of

105

Figure 4.6 Wavelength scans (absorption spectra) of quenched Fenton’s reaction
mixture. Wavelength scans were performed over a wavelength range of 400 nm to 750
nm. The “Pre-BA” wavelength scan represents a sample of the Fenton’s reaction mixture
prior to the benzoic acid chemical probe addition, but after completion of quenching.

The remainder of the wavelength scans represents F enton’s reaction mixture samples at 0
to 90 minutes following the addition of benzoic acid after the completion of quenching.
Polystyrene cuvettes with an optical pathlength of 10 mm were used.

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methylene blue dye test strip on test results by applying identical samples of unquenched
Fenton’s reaction solution to test strips aged 4 and 33 days, (B) the ability of 3% H202 to
cause bleaching of the methylene blue dye as compared to Milli-Q H20 and control (no
sample added) test strips, and (C) the affect of Milli-Q H2O pH on the methylene blue

dye test.

108

bleaching of these two test strips was not significantly different from the 24 hour old strip
tested with 40 uL of unquenched F enton’s reaction solution at 30 minutes of reaction
time (Figure 4.2D). In the second experiment (Figure 4.7B), 40 uL of 3% H202 was
tested for its ability to cause discoloration/bleaching in the methylene blue dye test as
compared with strips tested with 40 uL of Milli-Q H20 and no sample (control). Since
3% H202 did not appear to cause any discoloration/bleaching of the methylene blue dye,
the reaction that causes the discoloration/bleaching of the methylene blue dye is
independent of H202. It can therefore be assumed that the bleaching observed for the
methylene blue dye tests with unquenched Fenton’s reaction solutions was a result of the
presence of hydroxyl radicals rather than unreacted H202. In the third experiment (Figure
4.7C), the effect of Milli-Q H2O pH on the methylene blue dye test was evaluated by
testing 40 uL samples of Milli-Q H2O adjusted to pH 3.5, 5.4, and 9.4 by the addition of
0.5 M H2SO4 and/or 1M NaOH. Since no discoloration of methylene blue dye was
observed for Milli-Q H2O at any pH tested, methylene blue dye test results are not
influenced by sample pH. The bleaching observed for the methylene blue dye tests with
unquenched Fenton’s reaction solutions was a result of the presence of hydroxyl radicals

rather than an effect of an acidic pH.

4.4 Conclusions

A new procedure, the methylene blue dye test, qualitatively indicates the presence
of hydroxyl radicals through the immediate, distinct bleaching of methylene blue dye on
alpaper test strip. This method employs a simple procedure requiring inexpensive

materials, without the addition of competitive probe chemicals that potentially can

109

interfere with the reaction. A Fenton’s reaction with an Fe2+zH2O2 molar ratio of 1:20
generated hydroxyl radicals in Milli-Q water. The presence and absence of hydroxyl
radicals were determined prior to and following quenching of the F enton’s reaction with
10% sodium sulfite, respectively. Bleaching of methylene blue dye, due to the presence
of hydroxyl radicals in a sample, was indicated by a discoloration from a dark blue color
to an almost white color, concentrated at the point of application, with a dark blue
outline. A lack of bleaching indicated the absence of hydroxyl radicals in the sample.
The presence of hydroxyl radicals was verified by benzoic acid chemical probe
experiments with thin layer chromatography (TLC) and spectrophotometric wavelength
scans. The presence of hydroxyl radicals was indirectly determined by detection of
hydroxylated benzoic acids on TLC plates and a violet solution color with a peak
absorbance at a wavelength close to 520 nm. In experiments to test the performance of
the methylene blue dye test strips, age of the test strip, presence of H202, and sample pH

were determined to have no significant effect on the methylene blue dye test results.

110

4.5 References

I.

10.

ll.

12.

Satoh, A.Y.; Trosko, J .E.; Masten, S.J. Methylene Blue Dye Test for Rapid
Qualitative Detection of Hydroxyl Radicals Formed in a F enton’s Reaction Aqueous
Solution. Environ. Sci. T echnol. 2007, 41 (8), 2881-2887.

Lindsey, M.E.; Tarr, M.A. Quantitation of hydroxyl radical during Fenton oxidation
following a single addition of iron and peroxide. Chemosphere 2000, 41 (3), 409-417.

Yoshimura, Y.; Otsuka, K.; Uchiyarna, K.; Tanaka, H.; Tamura, K.; Ohsawa, K.;
Irnaeda, K. Detection of hydroxyl radicals with salicylic acid. Anal. Sci. 1989, 5 (2),
161-164.

Cao, Y.; Chu, Q.; Ye, J. Determination of hydroxyl radical by capillary
electrophoresis and studies on hydroxyl radical scavenging activities of Chinese
herbs. Anal. Bioanal. Chem. 2003, 376(5), 691-695.

Yang, X.-F.; Guo, X.-Q. Study of nitroxide-linked naphthalene as a fluorescence
probe for hydroxyl radicals. Anal. Chim. Acta 2001, 434 (2), 169-177.

Dutta, K.; Mukhopadhyay, S.; Bhattacharjee, S.; Chaudhuri, B. Chemical oxidation of
methylene blue using a F enton-like reaction. J. Hazard Mater. 2001, B84 (1), 57-71.

The Merck Index, 11'” Edition, Merck & Co., Inc.: Rahway, NJ, 1989, 954.

Kishore, K.; Guha, S.N.; Mahadevan, J .; Moorthy, P.N.; Mittal, J .P. Redox reactions
of methylene blue: A pulse radiolysis study. Radiat. Phys. Chem. 1989, 34 (4), 721-
727.

Sato, C.; Leung, S.W.; Bell, H.; Burkett, W.A.; Watts, R. J. Decomposition of
Perchloroethylene and Polychlorinated Biphenyls with Fenton's Reagent. In
Emerging Technologies in Hazardous Waste Management 111; Teddler, D. W.,
Pohland, F. G., Eds.; ACS Symposium Series 518; American Chemical Society:
Washington, DC, 1991; pp 343-356.

Trapido, M.; Goi, A. Degradation of nitrophenols with the F enton reagent. Proc.
Estonian Acad. Sci. Chem. 1999, 48(4), 163-173.

Dercova, K.; Branislav, V.; Tandlich, R.; Subova, L. F enton’s type reaction in
chemical pretreatment of PCBs. Chemosphere 1999, 39 (15), 2621-2628.

Bishop, D.F.; Stern, G.; Fleischrnan, M.; Marshall, L.S. Hydrogen peroxide catalytic

oxidation of refractory organics in municipal waste waters. Ind Eng. Chem. Proc.
Des. Dev. 1968, 7(1), 110-117.

111

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Mohamadin, A.M. Possible role of hydroxyl radicals in the oxidation of
dichloroacetonitrile by F enton-like reaction. J. Inorg. Biochem. 2001, 84 (1-2), 97-
1 05 .

Trapido, M. Tallinn Technical University, Tallinn, Estonia. Personal Communication,
2002.

Zhou, X.; Mopper, K. Determination of photochemically produced hydroxyl radicals
in seawater and freshwater. Mar. Chem. 1990, 30, 71-88.

Skoog, D.A.; Holler, F .J .; Nieman, T.A. Principles of Instrumental Analysis, 5th ed.;
Harcourt Brace & Company: Orlando, FL, 1998; pp 761-765.

Sherrna, J. Planar chromatography. Anal. Chem. 2002, 74 (12), 2653-2662.
Arnold, S.M.; Hickey, W.J.; Harris, R.F. Degradation of atrazine by Fenton’s
reagent: condition optimization and product quantification. Environ. Sci. Technol.

1995, 29(8), 2083-2089.

Pratap, K.; Lemley, A.T. Fenton electrochemical treatment of aqueous atrazine and
metolachlor. J. Agric. Food Chem. 1998, 46(8), 3285-3291.

Shriner, R.L.; Fuson, R.C.; Curtin, D.Y.; Morrill, T.C. The systematic Identification
of Organic Compounds, 6th ed.; Wiley: New York, 1980; pp. 348-350.

Esteves da Silva, J .C.G.; Machado, A.A.S.C.; Oliveira, C.J.S. Effect of pH on
complexation of Fe(III) with fulvic acids. Environ. Toxicol. Chem. 1998, 1 7(7),
1268-1273.

Wesp, E.F.; Brode, W.R. The absorption spectra of ferric compounds. I. The ferric
chloride-phenol reaction. J. Am. Chem. Soc. 1934, 56(5), 1037-1042.

112

Chapter 5

Toxicology Studies of Fenton’s Remediation in Milli-Q Water

5.1 Introduction

As a first step in evaluating Fenton’s reagent as a remediation process, it is
important to verify that the reaction of Fenton’s reagent with the solvent (in the absence
of 4,4'-dichlorobiphenyl (4,4'DCBP)) does not result in any toxic byproducts. If no toxic
response occurs as a result of a reaction of F enton’s reagent with the solvent, any toxicity
resulting from the F enton’s reagent remediation of 4,4'DCBP can be assumed to be
independent of the solvent. Additionally, this series of experiments allowed for the
development of a remediation procedure that later would be applied to 4,4'DCBP. The
three solvents investigated were Milli-Q H2O alone, 80/20 Milli-Q H2O/ACN (by
volume), and 50/50 Milli-Q H2O/ACN (by volume). In this chapter only F enton’s
reaction in Milli-Q H2O is presented. Reactions in the solvents 80/20 Milli-Q H2O/ACN
and 50/50 Milli-Q H2O/ACN are discussed in Chapter 6. Although 4,4'DCBP is
insoluble in water alone, since water constituted some part of the solvent used for
dissolving 4,4'DCBP in remediation, it was important to investigate whether Fenton’s
reagent remediation with water alone resulted in any toxicity. Three molar F e2+zH2O2
ratios, 1:5, 1:20, and 1:40, were investigated in this section of research to determine the
ratio that would be used for Fenton’s remediation of 4,4'DCBP. The toxicity of the
resulting final F enton’s remediation solutions was evaluated in rat liver epithelial cells
using a nongenotoxic bioassay that determines the in vitro modulation of gap junctional

intercellular communication (GJIC) as a measure of the epigenetic toxicity.

113

5.2 Experimental Section
5.2.1 Chemicals

For the Fenton’s remediation portion of this section of research the following
chemicals were used. Methylene blue dye (3,7-bis(dimethylamino)phenothiazin-5-ium
chloride) (97% purity) was purchased from Fluka (Buchs, Switzerland). Thirty-percent
hydrogen peroxide (H202) (unstabilized), iron (11) sulfate heptahydrate (FeSO4-7H2O)
(99% purity), and sodium sulfite (N a2SO3) (anhydrous, 98% purity) were purchased fi'om
Sigma—Aldrich (St. Louis, MO). Sulfuric acid (H2SO4) (96% purity) and sodium
hydroxide (N aOH) pellets (99% purity) were purchased from J .T. Baker (Phillipsburg,
NJ). Throughout this chapter and dissertation, Milli-Q water was obtained from a Milli-
Q Ultrapure Water Purification System (System Type ZMQS6VFOY) purchased from
Millipore Corp. (Bedford, MA).

For the toxicology portion of this section of research the following chemicals
were used. For cell culture, D-medium (Formula No. 78-5470 EG), Fetal Bovine Serum
(F BS), and Gentamicin were purchased from GIBCO Laboratories (Grand Island, NY).
Lucifer yellow CH, dilithium salt, was purchased from Molecular Probes Inc. (Eugene,
OR), ICN Biomedicals Inc. (Aurora, OH), and Sigma Chemical Co. (St. Louis, MO).
Formaldehyde solution (3 7%) for the GJIC bioassays was purchased fiom J .T. Baker

(Phillipsburg, NJ).

114

5.2.2 Methods
5.2.2.1 Fenton’s Remediation in Milli-Q Water

For each experiment, the initial concentration of H202 in the reaction mixture was
3 mM, which is within the range used by Trapido et al. (I) (1.0 to 10.0 mM). The initial
Fe2+ concentrations required for the Fe2+:H2O2 ratios 1:5, 1:20, and 1:40 were 0.6 mM,
0.15 mM, and 0.075 mM, respectively. These Fe2+ concentrations are within the range
used by Trapido et al. (1) (0.004 mM tol .0 mM). Throughout the remediation process,
the pH was adjusted/monitored using a 720A plus pH/ISE meter with an 8102 BNU Ross
Ultra Combination pH electrode (ThermoOrion, Beverly, MA).

The methylene blue (MB) dye test was performed during the F enton’s reaction to
verify the formation of hydroxyl radicals and during the quenching process to verify the
completion of quenching. MB dye test strips were prepared by the method of Satoh et al.
(2) as described in detail in Chapter 4. Forty rnicroliters of a liquid sample were placed
dropwise onto the center of the MB dyed section of a test strip, allowing for absorption
between drops. All MB dye tests were compared against a test strip tested with Milli-Q
water. The absence of bleaching of the MB dye indicated that no hydroxyl radicals were
present to the extent detectable by this qualitative test. In the quenching process of the
F enton’s reaction, the absence of bleaching signified that quenching was complete.
Bleaching of the MB dye, due to the presence of hydroxyl radicals in a sample, was
indicated by an immediate discoloration of the MB dye from a dark blue color to an
almost white color, concentrated at the point of application, with a dark blue outline.

For each F enton’s remediation experiment a 5.0 mM F eSO4 stock solution was

prepared from FeSO4-7H2O and Milli-Q water. The 5.0 mM FeSO4 stock solution was

115

then used to prepare 100 mL of a FeSO4 reaction solution with the pre-determined Fe2+
concentration required for the selected Fe2+zH2O2 ratio. A 150 mL glass beaker with a
stir bar was used as the reaction vessel. For optimal Fenton’s reaction efficiency, the
FeSO4 reaction solution was adjusted to pH 3 with 0.5 M H2SO4 and/or 1M NaOH. The
Fenton’s reaction was initiated by the addition of 3% H202, prepared from 30%
unstabilized H202 and Milli-Q water, to the reaction mixture to obtain an initial
concentration of 3 mM H2O2. Since stabilizing agents (hydroxyl radical scavengers) in
commercial H202 might affect the results (3), only H202 devoid of stabilizing agents was
used. In order to prevent localized reactions (that might occur when a small volume of
very concentrated solution is added to a reaction mixture), 3% H202 rather than 30%
H202 was used to initiate the reaction. To test, qualitatively, the production of hydroxyl
radicals during the reaction, MB dye tests were performed on the unquenched reaction
mixture at 15, 30, and/or 60 minutes of reaction.

After 60 minutes, the F enton’s reaction was quenched with a 10% Na2SO3
solution (w/v), prepared from Na2SO3 and Milli-Q water. Trapido et al. (I, 4)
recommended quenching by the addition of 2 to 3 drops of 10% Na2SO3 solution for
every 10 mL of reaction mixture. A 0.5 mL glass pipette was used to administer the
drops to the reaction mixture. To verify, qualitatively, that quenching was complete, 5
minutes after the addition of the 10% Na2SO3 solution, a MB dye test was performed. An
additional aliquot of 10% Na2SO3 solution was added to the reaction mixture, and the
MB dye test was repeated until no discoloration of the MB dye was observed (quenching
was complete). To aid in the removal of iron fiom the reaction mixture by precipitation

of Fe“, following quenching, the reaction mixture was adjusted to pH 9 with 0.5 M

116

H2SO4 and/or 1M NaOH. If a large amount of precipitate in the reaction mixture was
observed, the solution was centrifuged at 1200 rpm for 10 minutes and the supernatant
was retained for filtration. The reaction mixture or supernatant was then filtered through
a 1.0 um glass fiber filter, using a vacuum filtration unit, into a 1000 mL Erlenmeyer
side-arm filtration flask. Milli-Q water was used for rinsing during filtration only if the
reaction mixture touched the sides of the firnnel or there was residue in the reaction
vessel. The filtered reaction mixture was transferred to a clean 150 mL glass beaker
containing a stir bar. To neutralize the reaction mixture pH for the cells in the toxicology
bioassays, the filtered solution was adjusted to pH 7 with 0.5 M H2SO4 and/or 1M NaOH.
This final solution was transferred to an amber, 120 mL Boston round bottle with a TFE

closure and was refrigerated at 4 °C until toxicology bioassays were performed.

5.2.2.2 Cell Culture Techniques

WB-F344 rat liver epithelial cells were obtained from Dr. J. W. Grisham and Dr.
M. S. Tsao of the University of North Carolina (Chapel Hill, NC) (5). This cell line was
selected because it is a diploid, nontumorigenic cell line originating from a strain of rat
that has been used for toxicological/cancer studies of numerous chemicals, thereby
allowing for a source of comparison (5). Since 70% of the chemicals that are carcinogens
are liver carcinogens and the liver is the “first pass” organ for ingested toxins, liver cells
are important for toxicological/cancer studies (6). Furthermore, the WB-F344 cell line
was designed for in vitro assays to match the many in vivo tumor promotion assays that

had been done in rat liver, specifically, in the Fischer 344 rat.

117

The cell culture techniques performed were similar to those described by Hemer
et al. (5) and Luster-Teasley et al. (7). Cells were cultured in 150 cm2 sterile, treated,
polystyrene cell culture flasks (Corning Inc., Corning, NY) in 25 mL of D-medium
containing 5% Fetal Bovine Serum (FBS) and 0.2% Gentamicin. The cells were
incubated at 37 °C in a water-j acketed IR Autoflow Automatic CO2 incubator (NU AIRE,
Inc., Plymouth, MN) in a humidified atmosphere with 5% CO2 and 95% air. The time
required for cell grth confluency was about two days. The confluent culture was split
and transferred every other day into a new 150 cm2 culture flask with new medium
mixture. In addition, from 150 cm2 flasks of confluent cells, cultures were prepared for
the bioassays in 35 rmn diameter, sterile, treated polystyrene cell culture dishes (Corning
Inc., Corning, NY) with 2 mL of D-medium supplemented with 5% F BS. The cultures

for the bioassays were incubated under the same conditions as the aforementioned flasks.

5.2.2.3 In Vitro Bioassay for GJIC

The bioassays of GJIC were performed on confluent cell cultures (usually 2 days
of growth) grown in 35 mm diameter culture dishes (as described in the preceding
section). The scrape-loading/dye transfer (SL/DT) procedure for determining the GJIC
was adapted from the method described by El-Fouly et al. (8) and is described in detail
by Hemer et al. (5 ). A detailed description of the spread of Lucifer yellow dye from the
scrape to neighboring cells can be found in Wilson et al. (9).

For reasons to be discussed later in this chapter, which determined that further
toxicology studies were unnecessary, only dose-response GJIC bioassays were

performed. Chemical treatments (reaction mixture solution), controls (no close), and

118

vehicle controls were performed in triplicates. Doses were applied directly to the dishes
of confluent cell cultures. The final reaction mixture solution, that was stored at 4 °C,
was warmed to room temperature prior to use in the bioassay. All GJIC bioassay results
assumed that the filtration process performed during the F enton’s remediation experiment
removed no toxic chemicals generated during the reaction. Since the exact molecular
weight and concentrations of the reaction mixture solutions were unknown, GJIC
bioassay doses were investigated in terms of volumes rather than concentrations. For the
dose-response experiments, confluent cells were exposed to dose volumes of 100, 150,
200, 250, and 300 uL of reaction mixture solution with incubation times of 30 minutes
(for each volume) and 2 hours (for the highest tested volume). Vehicle controls for the
GJIC bioassays were performed using Milli-Q water and were incubated for the same
incubation times. Vehicle controls were dosed with a volume corresponding to the
largest volume of reaction mixture tested in the treatment dishes. Following incubation,
the GJIC was determined by the SL/DT procedure. All culture dishes were examined
within 24 hours of completion of the experiment. Each culture dish of cells was digitally
photographed such that the observed scrape spanned the fill] horizontal width of the
picture. A COHU High Performance Color CCD camera (Cohu, Inc., San Diego, CA)
with a magnification of 200x under a Nikon TE300 Eclipse Inverted Microscope (Nikon
Corp., Japan) with a Nikon HB-10103AF Super High Pressure Mercury 100W lamp
(Nikon Corp., Japan) was used.

The fluorescence of the Lucifer yellow dye was used to determine the distance the
dye traveled perpendicular to the scrape. The distance of dye travel was indicative of the

level of GJIC within the culture. Quantitative analysis of the distance of dye spread was

119

performed using NucleoTech GelExpert software (Nucleotech Corp., Hayward, CA).

The distance of dye spread was measured in terms of the area of dye spread by tracing
manually via free object quantification the area of farthest visible fluorescence. Since the
width of the photographed section was the same for every culture dish, measuring the
area of dye spread was equivalent to measuring the distance of dye spread perpendicular
to the scrape. The area of dye spread for each chemical treatment dish was compared to a
control group of cells that were exposed to Milli-Q water only (vehicle controls) under
the same assay as the treated cells. For each chemically treated dish, the fraction of the
control was calculated as the area of dye spread in the treated dish divided by the average
area of dye spread in the triplicate set of vehicle control dishes. The results for each set
of chemically treated triplicates were reported as an average fraction of the control (FOC)
i standard deviation (SD) determined at the 95% confidence interval (CI).

The level of GJIC in cells exposed to the chemical treatment was assessed by the
decrease in communication of the cells as compared to the vehicle control groups. A
decrease in FOC corresponds directly to a decrease in GJIC (where the doses are not
cytotoxic). Interpretations of GJIC results were consistent with Luster-Teasley et al (7)
and Hemer et al. (5). Complete communication (100%) between the cells is identified as
a F OC value of 1.0 as seen in the vehicle control. A FOC value greater than 0.9 is
difficult to statistically distinguish fiom the vehicle controls. FOC values between 0.9
and 0.5 indicate partial inhibition of GJIC. A FOC value less than or equal to 0.5 is
indicative of a significant amount of inhibition of GJIC, since this would be
representative of communication levels that are 50% or less than the normal

communication levels. FOC values between 0.3 and 0.0 are representative of complete

120

inhibition of GJIC. A F OC value of 0.3 is usually used to represent complete inhibition,
as it corresponds to the width of a single row of cells with no dye spreading beyond its
boundaries (5). Controls, which received no dose of chemical or Milli-Q water, were
performed for each experiment as a means of evaluating a normal level of GJIC and the
overall “health” of the cells. By performing a t-test for each experiment, it was found
that the areas of dye spread for the control dishes did not vary significantly from the areas
for the vehicle controls at a 95% CI. Therefore, it could be concluded that the Milli-Q
water, at the volume tested, was not a significant source of inhibition in the experiments.
Statistical analyses were performed by means of the t-test and one-way analysis of

variance (ANOVA) to compare the chemical treatment results and vehicle control results.

5.3 Results and Discussion
5.3.1 Fenton’s Remediation in Milli-Q Water

For each molar Fe2+zH2O2 ratio investigated, during the Fenton’s remediation
experiments, prior to quenching, no significant change in solution pH (~ 3.0) and
temperature (23.0 °C) occurred during the 60 minute reaction period. Although for the
Fe2+zH2O2 ratios 1:20 and 1:40 the unquenched Fenton’s reaction mixture remained clear
and colorless in appearance during the reaction period, for the F e2+zH2O2 ratio 1:5 the
solution appeared very light orange and slightly cloudy. Figures 5.1 and 5.2 present the
MB dye test results for F enton’s remediation in Milli-Q water with Fe2+zH2O2 ratios 1:20
and 1:40, respectively. For each of the Fe2+:H2O2 ratios, MB dye tests indicated the
presence of hydroxyl radicals during the reaction period (unquenched) by an immediate

bleaching of the MB dye. Similar degrees of bleaching were observed for the reaction

121

Figure 5.1 Fenton’s remediation in Milli-Q water with a Fe2+zH2O2 ratio of 1:20.
Methylene blue dye test results for (A) control (no sample added); (B) Milli-Q water;
unquenched F enton’s reaction mixture at (C) 15 minutes and (D) 30 minutes of reaction;
and Fenton’s reaction mixture quenched with (E) 30 drops and (F) 35 drops of 10%
Na2SOg. All methylene blue dye tests were performed using 40 ILL samples.

122

123

 

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Figure 5.2 Fenton’s remediation in Milli-Q water with a Fe2+zH2O2 ratio of 1:40.
Methylene blue dye test results for (A) control (no sample added); (B) Milli-Q water;
unquenched Fenton’s reaction mixture at (C) 15 minutes, (D) 30 minutes, and (E) 60
minutes of reaction; and F enton’s reaction mixture quenched with (F) 33 drops of 10%
Na2SO3. All methylene blue dye tests were performed using 40 uL samples.

124

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125

times tested for each of the F e2+:H2O2 ratios. Although the bleaching appeared to be
similar for F e2+:H2O2 ratios of 1:5 and 1:20, the bleaching appeared to be significantly
greater for the Fe2+zH2O2 ratio of 1:20 (Figures 5.1C and D) than for 1:40 (Figures 5.2C,
D, and E), therefore suggesting a greater production of hydroxyl radicals with 1:5 and
1:20 than 1:40. One possible explanation for this observation is that the amount of
hydroxyl radicals produced will vary with the Fe2+zH2O2 ratio, because the chemical
species present will vary. In addition, since Fe2+ acts as a catalyst in the Fenton’s
reaction, the rate of hydroxyl radical production will also be affected by the F e2+zH2O2
ratio. For all the experiments, the MB dye test control strip (no sample added) was
homogeneously dark blue in color (Figures 5.1A and 5.2A) and no bleaching or
discoloration was observed for the MB dye test performed with Milli-Q water (Figures
5.1B and 5.2B).

The Fenton’s reaction was quenched after 60 minutes. For each F e2+:H2O2 ratio,
the pH of the reaction mixture increased from approximately 3.0 to 7.7. The temperature
remained constant at approximately 23.0 °C. The color of the reaction mixture changed
from colorless to varying intensities of orange dependent on the Fe2+zH2O2 ratio. For the
Fe2+zH2O2 ratios of 1:5, 1:20, and 1:40, the color changed to dark orange (rust-like), light
orange, and very light orange, respectively. This color change to orange can be attributed
to the more basic pH of the reaction mixture during the quenching process, resulting in
the conversion of iron from a hydrated ferrous form to a colloidal ferric form and the
formation of ferric hydroxide (10, 11, 12). To verify, qualitatively, that quenching was
complete, a MB dye test was performed. Additional 10% Na2303 solution was added to

the reaction mixture and the MB dye test was repeated until no discoloration of the MB

126

dye was observed (quenching was complete). For the Fe2+zH2O2 ratios of 1:20 and 1:40,
the MB dye test results of the quenched F enton’s reaction mixtures are shown in Figures
5.1 E and F, and Figure 5.2F, respectively. Complete quenching of the reaction was
achieved for the Fe2+zH2O2 ratios of 1 :5, 1:20, and 1:40 by the addition of a total of 35
drops (1.55 mL), 35 drops (1.44 mL), and 33 drops (1.27 mL) of 10% Na2S03 solution,
respectively.

To aid in the removal of iron from the reaction mixtures by precipitation of F e3+,
following quenching, the reaction mixture was adjusted to approximately pH 9.
Following this pH adjustment, the solution remained clear and colorless with very fine
rust colored (light orange) particles in suspension. The amount of precipitate for the
Fe2+:H2O2 ratio 1:20 was slightly greater than that observed for 1:40, but the amount of
precipitate was not great enough for either solution to require centrifirgation. However,
since the amount of precipitate observed for 1:5 was significantly greater than observed
for either 1:20 or 1:40, this solution was centrifuged at 1200 rpm for 10 minutes and the
supernatant was retained for filtration. The reaction mixture or supernatant was then
filtered and 5 mL of Milli-Q water was used for rinsing during the filtration process. For
each solution, the filtrate appeared clear and colorless with no precipitate. To neutralize
the reaction mixture pH for the cells in the toxicology bioassays, each filtered solution
was adjusted to pH 7. Following adjustment of the filtrate to pH 7, each solution
remained clear and colorless with no precipitate. These final reaction mixture solutions

were stored at 4 °C.

127

5.3.2 In Vitro Bioassay for GJIC

Prior to and following warming to room temperature, the final reaction mixture
solutions were clear and colorless with no precipitate. Figures 5.3, 5.4, and 5.5 present
30 minute incubation dose-response GJIC bioassay results for a volume range of 0 to 300
uL of final reaction mixture solutions corresponding to the F e2+2H2O2 ratios 1:5, 1:20,
and 1:40, respectively. For each of the Fe2+zH2O2 ratios, no inhibition of GJIC was
observed after 30 minutes of incubation time. As shown in Table 5.1, for the highest
tested volume (300 uL), no inhibition of GJIC was observed at 30 minutes and 2 hours of

incubation.

Table 5.1 300 uL GJIC assay results for 30 minutes and 2 hour incubation times

 

 

Fe2+zH202 30 minutes of incubation 2 hours of incubation
Ratio (FOCiSD) (FOCiSD)
l :5 1.01i0.02 1.02i0.01
1:20 l.00:t0.02 l.00i0.02
1:40 1 .011’0.03 l.00i0.01

 

 

 

 

 

Since no inhibition of GJIC was observed for the dose-response GJIC bioassay
results for incubation times of 30 minutes (0 to 300 uL) and 2 hours (300 uL), it was
decided that cytoxicity, time-response, and time of recovery bioassays were not required.
In addition, since the final reaction mixture solutions produced from a 1 hour Fenton’s
reagent reaction (for the selected Fe2+zH2O2 ratios) did not produce any inhibition of
GJIC in the dose-response bioassays, it was decided that the investigation of longer

reaction times (5 hours and 24 hours) were unnecessary. Although some researchers

128

 

1.00 W ..

 

0.75 F a

GJIC
(Fraction of the Control)

0.50 - q

 

 

 

0.25 l" -I
0.00 I l L l I I i
0 50 100 150 200 250 300

Volume of Sample Added (uL)

Figure 5.3 Dose-response GJIC bioassay results after a 30 minute incubation time for a
volume range of 0 to 300 uL of a solution resulting from Fenton’s reagent remediation
with only Milli-Q water solvent (no PCB), a Fe2+zH2O2 ratio of 1:5, and 1 hour reaction
time. Each data point is representative of the results for a set of chemically treated
triplicates reported as an average FOC i standard deviation determined at the 95%
confidence interval.

129

 

 

 

 

 

I I I I I W I
1.00 4——§—o—-—O—I 4
‘5
5 0.75 - -
0
2 §
3 “5
c 0.50 ..
,3 r
0
s
a
0.25 - ..
0.00 l 1 I I I l
0 50 100 150 200 250 300

Volume of Sample Added (uL)

Figure 5.4 Dose-response GJIC bioassay results after a 30 minute incubation time for a

volume range of 0 to 300 uL of a solution resulting from F enton’s reagent remediation
with only Milli-Q water solvent (no PCB), a F e2+:H2O2 ratio of 1:20, and 1 hour reaction
time. Each data point is representative of the results for a set of chemically treated
triplicates reported as an average F OC i standard deviation determined at the 95%
confidence interval.

130

 

 

1.00 M -

0.75 - .-

GJIC
(Fraction of the Control)

0.50 - _

 

 

 

0.25 - -
0.00 I I I I I i 4 I
O 50 100 150 200 250 300
Volume of Sample Added (ILL)

Figure 5.5 Dose-response GJIC bioassay results after a 30 minute incubation time for a
volume range of 0 to 300 ML of a solution resulting from Fenton’s reagent remediation
with only Milli-Q water solvent (no PCB), a Fe2+:H2O2 ratio of 1:40, and 1 hour reaction
time. Each data point is representative of the results for a set of chemically treated
triplicates reported as an average FOC 1- standard deviation determined at the 95%
confidence interval.

131

have used reaction times of up to 1 week (13, 14), a 1 hour (3600 seconds) reaction time

is consistent with that used by Trapido et al. (1).

5.4 Conclusions

Although 4,4'DCBP is insoluble in water alone, since water (Milli-Q) constituted
some part of the solvent used for dissolving 4,4'DCBP in remediation, it was important to
investigate whether F enton’s reagent remediation with water alone resulted in any
toxicity. For the final reaction mixture solutions corresponding to the Fe2+zH2O2 ratios
1:5, 1:20, and 1:40, no inhibition of GJIC was observed for the dose-response GJIC
bioassay for incubation times of 30 minutes (0 to 300 uL) and 2 hours (300 uL). Hence,
Fenton’s reagent remediation with water alone can be expected to not result in any
toxicity, and any toxicity resulting from the Fenton’s reagent remediation of 4,4'DCBP
can be assumed to be independent of the influence of water. The remediation procedure
developed in this series of experiments was later applied to the Fenton’s remediation of
4,4'DCBP. Based on the results of Fenton’s remediation in Milli-Q water, an Fe2+:H2O2
ratio of 1:20 was selected for Fenton’s remediation of 4,4'DCBP, since it appeared to
result in greater production of hydroxyl radicals than 1:40 and resulted in less F e3 +
precipitate prior to filtration than 1:5. This selected Fe2+zH2O2 ratio is consistent with the
optimal ratio for remediation recommended by Trapido et al. (1). In addition, it is
reported that the formation of hydroxyl radicals by the Fenton’s reagent reaction occurs
best when the molar ratio of Fe2+:H2O2 is kept at a value of 1:15 at a pH of 3 (15), which

is close to 1:20.

132

5.5 References

1.

Trapido, M.; Goi, A. Degradation of nitrophenols with the Fenton reagent. Proc.
Estonian Acad Sci. Chem. 1999, 48(4), 163-173.

Satoh, A.Y.; Trosko, J .E.; Masten, S.J. Methylene Blue Dye Test for Rapid
Qualitative Detection of Hydroxyl Radicals Formed in a Fenton’s Reaction Aqueous
Solution. Environ. Sci. T echnol. 2007, 41 (8), 2881-2887.

Mohamadin, A.M. Possible role of hydroxyl radicals in the oxidation of
dichloroacetonitrile by Fenton-like reaction. J. Inorg. Biochem. 2001, 84 (1-2), 97-
105.

Trapido, M. Tallinn Technical University, Tallinn, Estonia. Personal Communication,
2002.

Hemer, H.A.; Trosko, J .E.; Masten, S.J. The Epigenetic Toxicity of Pyrene and
Related Ozonation Byproducts Containing an Aldehyde Functional Group. Environ.
Sci. T echnol. 2001, 35 (17), 3576-3583.

Trosko, J .E. Michigan State University, East Lansing, MI. Personal Communication,
August 2003.

Luster-Teasley, S.L.; Yao, J .J .; Hemer, H.A.; Trosko, J .E.; Masten, S.J. Ozonation of
Chrysene: Evaluation of Byproduct Mixtures and Identification of Toxic Constituent.
Environ. Sci. T echnol. 2002, 36 (5), 869-876.

El-Fouly, M.H.; Trosko, J.E.; Chang, C.C. Scrape-Loading and Dye Transfer: A rapid
and simple technique to study gap junctional intercellular communication. Exp. Cell
Res. 1987, 168, 422-430.

Wilson, M.R.; Close, T.W.; Trosko, J.E. Cell Population Dynamics (Apoptosis,
Mitosis, and Cell-Cell Communication) during Disruption of Homeostasis. Exp. Cell
Res. 2000, 254, 257-268.

10. Lindsey, M.E.; Tarr, M.A. Quantitation of hydroxyl radical during Fenton oxidation

following a single addition of iron and peroxide. Chemosphere 2000, 41 (3), 409-417.

11. Arnold, S.M.; Hickey, W.J.; Harris, RF. Degradation of atrazine by Fenton’s

reagent: condition optimization and product quantification. Environ. Sci. Technol.
1995, 29(8), 2083-2089.

12. Pratap, K.; Lemley, A.T. Fenton electrochemical treatment of aqueous atrazine and

metolachlor. J. Agric. Food Chem. 1998, 46 (8), 3285-3291.

133

13. Sedlak, D.L.; Andren, A.W. Aqueous-Phase Oxidation of Polychlorinated Biphenyls
by Hydroxyl Radicals. Environ. Sci. T echnol. 1991, 25(8), 1419-1427.

14. Dercova, K.; Branislav, V.; Tandlich, R.; Subova, L. Fenton's Type Reaction and
Chemical Pretreatment of PCBs. Chemosphere 1999, 39(15), 2621-2628.

15. Dutta, K.; Mukhopadhyay, S.; Bhattacharjee, S.; Chaudhuri, B. Chemical oxidation of
methylene blue using a F enton-like reaction. J. Hazard. Mater. 2001, 384(1), 57-71.

134

Chapter 6

Toxicology Studies of Fenton’s Remediation of Potential Solvents
for 4,4’-Dichlor0biphenyl

6.1 Introduction

A combination of Milli-Q H20 and acetonitrile (ACN) was selected as the solvent
for dissolving 4,4’-dichlorobiphenyl (4,4'DCBP) for use in the Fenton’s remediation
experiments. Evaluation of whether Fenton’s reagent remediation in water alone resulted
in any toxicity is discussed in Chapter 5. Acetonitrile was added as part of the solvent to
increase the solubility of the 4,4’DCBP. It has been shown that the presence of the
acetonitrile does not inhibit the Fenton’s reaction, as it is not appreciably reactive with
hydroxyl radicals (1 ), and in toxicology studies it does not affect GJIC at low
concentrations (2). In this chapter, Fenton’s remediation of the solvents 80/20 Milli-Q
H2O/ACN and 50/50 Milli-Q H2O/ACN (by volume) were investigated to determine the
toxicological effect of ACN in combination with water as a solvent in remediation. If no
toxic response occurs as a result of a reaction of Fenton’s reagent with the solvent, any
toxicity resulting from the Fenton’s reagent remediation of 4,4'DCBP can be assumed to
be independent of the solvent.

Based on the results of Fenton’s remediation in Milli-Q water, discussed in detail
in Chapter 5 , an Fe2+zH2O2 ratio of 1:20 was selected for each Fenton’s remediation
experiment. Since Chapter 4 evaluated the use of the Methylene Blue (MB) Dye Test for
Fenton’s reactions in Milli-Q water only, further experiments were performed to study
the MB dye test in the presence of ACN and Milli-Q H2O/ACN. The toxicity of the

resulting final Fenton’s remediation solutions was evaluated in rat liver epithelial cells

135

using a nongenotoxic bioassay that determines the in vitro modulation of gap junctional

intercellular communication (GJIC) as a measure of the epigenetic toxicity.

6.2 Experimental Section
6.2.1 Chemicals

For the Fenton’s remediation portion of this section of research the following
chemicals were used. Methylene blue dye (3,7-bis(dimethylamino)phenothiazin—5-ium
chloride) (97% purity) was purchased from Fluka (Buchs, Switzerland). Acetonitrile
(99.8% purity) was purchased from EM Science (Gibbstown, NJ). Thirty-percent
hydrogen peroxide (H202) (unstabilized), iron (11) sulfate heptahydrate (FeSO4-7H2O )
(99% purity), and sodium sulfite (N a2SO3) (anhydrous, 98% purity) were purchased fiom
Sigma—Aldrich (St. Louis, MO). Sulfuric acid (H2SO4) (96% purity) and sodium
hydroxide (N aOH) pellets (99% purity) were purchased fiom J .T. Baker (Phillipsburg,
NJ). Throughout this chapter and dissertation, Milli-Q water was obtained from a Milli-
Q Ultrapure Water Purification System (System Type ZMQS6VFOY) purchased from
Millipore Corp. (Bedford, MA).

For the toxicology portion of this section of research the following chemicals
were used. For cell culture, D-medium (Formula No. 78-5470 EG), Fetal Bovine Serum
(F BS), and Gentamicin were purchased from GIBCO Laboratories (Grand Island, NY).
Lucifer yellow CH, dilithium salt, was purchased fiom Molecular Probes Inc. (Eugene,
OR), ICN Biomedicals Inc. (Aurora, OH), and Sigma Chemical Co. (St. Louis, MO).
Formaldehyde solution (37%) for the GJIC bioassays was purchased from J .T. Baker

(Phillipsburg, NJ).

136

6.2.2 Methods
6.2.2.1 Preliminary Methylene Blue Dye Tests

Two experiments were performed to evaluate the MB dye test in the presence of
ACN and Milli-Q H2O/ACN. MB dye test strips were prepared by the method of Satoh
et al. (3) as described in detail in Chapter 4. Liquid sample was placed dropwise onto the
center of the MB dyed section of a test strip, allowing for absorption between drops. In
the first experiment, 40 uL of 100% ACN, 80/20 Milli-Q H2O/ACN solvent, and 50/50
Milli-Q H2O/ACN were tested on separate test strips for their ability to cause
discoloration/bleaching in the MB dye test as compared with strips tested with 40 IrL of
Milli-Q H20 and no sample (control). In the second experiment, the effect of the pH of
the 80/20 Milli-Q H2O/ACN solvent on the MB dye test was evaluated by testing 40 uL
samples of 80/20 Milli-Q H2O/ACN solvent adjusted to a variety of pH values by the

addition of 0.5 M H2SO4 and/or 1M NaOH.

6.2.2.2 Fenton’s Remediation of 80/20 and 50/50 Milli-Q Water/Acetonitrile

Based on the results of Fenton’s remediation in Milli-Q water (Chapter 5), an
Fe2+:H2O2 molar ratio of 1 :20 was selected for the Fenton’s remediation of 4,4'-
dichlorobiphenyl (4,4'DCBP) and therefore Fenton’s remediation of the solvents 80/20
Milli-Q H2O/ACN and 50/50 Milli-Q H2O/ACN. This selected Fe2+zH2O2 ratio is
consistent with the optimal ratio for remediation recommended by Trapido et a1. (4).
Furthermore, it is reported that the formation of hydroxyl radicals by the Fenton’s reagent
reaction occurs best when the molar ratio of Fe2+zH2O2 is kept at a value of 1:15 at a pH

of 3 (5), which is close to 1:20. The initial concentrations of Fe2+ and H202 in the

137

reaction mixtures were 0.15 mM and 3 mM, respectively, which are within the ranges
previously used by Trapido et al. (4). The volume of 5.0 mM FeSO4 and 3% H202 added
to the reaction mixture was calculated to reflect the assumption that this addition caused a
significant change in the overall reaction mixture volume.

The amount of water added by the addition of 5.0 mM FeSO4, pH adjustment,
addition of 3% H202, quenching, and rinsing of the filtration funnel was monitored
throughout the experiment, and an adjusted total H2O/ACN ratio was calculated at the
completion of the experiment. In this calculation, the volumes of 5.0 mM F e804, 3%
H202, 10% Na2SO3, 0.5 M H2SO4, and 1M NaOH added were assumed to be 100%
water. The total H2O/ACN ratio suggested the degree of dilution of the sample that
occurred during the experiment. In each experiment, this ratio was maintained as
constant as possible to allow for comparison of the results. The pH and temperature were
monitored throughout the experiments.

MB dye tests were performed during the Fenton’s reaction to verify the formation
of hydroxyl radicals and during the quenching process to verify the completion of
quenching (absence of hydroxyl radicals). Forty microliters of liquid sample were placed
dropwise onto the center of the MB dyed section of a test strip, allowing for absorption
between drops. For the remediation of each solvent investigated, MB dye tests were
compared against a test strip tested with 80/20 Milli-Q H2O/ACN or 50/50 Milli-Q
H2O/ACN, respectively. The presence of hydroxyl radicals in a reaction mixture was
indicated in the MB dye test by an immediate concentrated white discoloration
(bleaching) characteristic to the solvent being used. MB dye tests indicating the absence

of hydroxyl radicals in a reaction mixture were identified by a resemblance to the MB

138

dye test results for the respective Milli-Q H2O/ACN solvent alone and a lack of
bleaching.

For each Fenton’s remediation experiment, the procedure started with the
preparation of either the 80/20 Milli-Q H2O/ACN or 50/50 Milli-Q H2O/ACN solvent.
The volume of solvent prepared as the initial reaction mixture was calculated as the
predicted initial reaction mixture volume for Fenton’s remediation of 4,4'DCBP. Since
in previous toxicology studies (Chapter 3) the final concentration of the test solution was
2 mM 4,4'DCBP, the initial reaction mixture volume for F enton’s remediation of
4,4'DCBP was predicted (based on calculations with 6 mg of 4,4'DCBP) to be close to 20
mL. Therefore, for Fenton’s remediation of 80/20 Milli-Q H2O/ACN and 50/50 Milli-Q
H2O/ACN solvent, the initial reaction mixture consisted of 20 mL of the respective
solvent. A 50 mL centrifuge tube with a vaned tube stir bar was used as the reaction
vessel. A 5 .0 mM FeSO4 stock solution was prepared from FeSO4°7H2O and Milli-Q
water. The 5.0 mM FeSO4 stock solution was then added to the reaction mixture at the
calculated volume to achieve a final concentration of 0.15 mM Fez)". For optimal
Fenton’s reaction efficiency, the reaction mixture was adjusted to pH 3 with 0.5 M H2SO4
and/or 1M NaOH. The pH was adj usted/monitored using a 720A plus pH/ISE meter with
an 8102 BNU Ross Ultra Combination pH electrode (ThermoOrion, Beverly, MA).

The Fenton’s reaction was initiated by the addition of 3% H202, prepared from
30% unstabilized H202 and Milli-Q water, to the reaction mixture to obtain an initial
concentration of 3 mM H202. Since stabilizing agents (hydroxyl radical scavengers) in
commercial H202 might affect the results (6), only H2O2 devoid of stabilizing agents was

used. In order to prevent localized reactions (that might occur when a small volume of

139

very concentrated solution is added to a reaction mixture), 3% H202 rather than 30%
H2O2 was used to initiate the reaction. To test, qualitatively, the production of hydroxyl
radicals during the reaction, MB dye tests were performed on the unquenched reaction
mixture at 15, 30, and 60 minutes of reaction. In addition, MB dye tests were performed
on 40 uL sarnples of Milli-Q water, and 80/20 Milli-Q H2O/ACN or 50/50 Milli-Q
H2O/ACN solvent.

After 60 minutes, the Fenton’s reaction was quenched with a 10% aqueous
solution of Na2SOg. Trapido et al. (4, 7) recommended quenching by the addition of 2 to
3 drops of 10% Na2803 solution for every 10 mL of reaction mixture. A 0.5 mL glass
pipette was used to administer the drops to the reaction mixture. To verify, qualitatively,
that quenching was complete, 5 minutes after the addition of the 10% Na2803 solution, a
MB dye test was performed. An additional aliquot of 10% Na2803 solution was added to
the reaction mixture, and the MB dye test was repeated until an absence of hydroxyl
radicals in the reaction mixture was indicated (quenching was complete). To aid in the
removal of iron from the reaction mixture by precipitation of Fe”, following quenching,
the reaction mixture was adjusted to pH 9 with 0.5 M H2SO4 and/or 1 M NaOH. If a
large amount of precipitation in the reaction mixture was observed, the solution was
centrifuged at 1200 rpm for 10 minutes and the supernatant was retained for filtration.
The reaction mixture or supernatant was then filtered through a 1.0 pm glass fiber filter,
using a vacuum filtration unit, into a large culture tube inserted into a 1000 mL
Erlenmeyer side-arm filtration flask. Milli-Q water was used for rinsing during filtration
only if the reaction mixture touched the sides of the funnel or there was residue in the

centrifirge tube. The filtered reaction mixture was transferred to a clean 50 mL centrifuge

140

tube containing a vaned tube stir bar. To neutralize the reaction mixture pH for the cells
in the toxicology bioassays, the filtered solution was adjusted to pH 7 with 0.5 M H2SO4
and/or 1M NaOH. This final solution was transferred to an amber, 60 mL Boston round
bottle with a TF E closure and was refiigerated at 4 °C until the toxicology bioassays were

performed.

6.2.2.3 Cell Culture Techniques
The cell culture techniques performed were identical to those described in

Chapter 5.2.2.2 for toxicology studies of Fenton’s remediation in Milli-Q water.

6.2.2.4 In Vitro Bioassay for GJIC

For reasons to be discussed later in this chapter, based on results which indicated
that further toxicology studies were unnecessary, only dose-response GJIC bioassays
were performed. Dose-response GJIC bioassays were performed in a manner similar to
that used for the resulting solution fi'om Fenton’s remediation in Milli-Q water (Chapter
5.2.2.3). The GJIC bioassays were performed on confluent WB-F344 cell cultures
(usually 2 days of growth) grown in 35 mm diameter culture dishes (as described in
Chapter 5.2.2.2) with 2 mL of D-medium supplemented with 5% FBS. Chemical
treatments (reaction mixture solution), controls (no dose), and vehicle controls were
performed in triplicates. All GJIC bioassay results assumed that the filtration process
performed during the Fenton’s remediation experiment removed no toxic chemicals
generated during the reaction. Since the exact molecular weight and concentration of the

reaction mixture solutions were unknown, GJIC bioassay doses were investigated in

141

terms of volumes rather than concentrations. Since a slight inhibition of GJIC occurs at a
final ACN concentration of 2.0% in the cell culture medium (corresponding to 40 (IL of
ACN), all GJIC bioassays were conducted with a final ACN concentration of 1.5% or
less (corresponding to 30 uL of ACN or less) (2). In the dose-response bioassay, if the
same maximum volume (300 uL) of the resulting solution from Fenton’s remediation in
Milli-Q water was used for the resulting solutions from F enton’s remediation of 80/20
Milli-Q H2O/ACN and 50/50 Milli-Q H2O/ACN, this would represent dose volumes of
60 uL and 150 uL of ACN, respectively. Therefore, the maximum dose volumes that
could be tested for the resulting solutions fi'om Fenton’s remediation of 80/20 Milli-Q
H2O/ACN and 50/50 Milli-Q H2O/ACN were 150 uL and 60 uL, respectively. The
volumes tested in the dose-response GJIC bioassay for the resulting solution from
Fenton’s remediation of 80/20 Milli-Q H2O/ACN were 20, 40, 80, 100, and 150 uL. The
volumes tested in the dose-response GJIC bioassay for the resulting solution from

F enton’s remediation of 50/50 Milli-Q H2O/ACN were 10, 20, 30, 40, 50, and 60 uL.
For each of the solutions investigated, the bioassays were incubated for 30 minutes for
each volume tested and for 2 hours for the highest volume tested.

Vehicle controls for the GJIC bioassays were performed using the respective
Milli-Q H2O/ACN solvent and were incubated for the same incubation times. To be
conservative, either the 80/20 or 50/ 50 Milli-Q H2O/ACN solvent was used for the
vehicle controls instead of a solution based on the adjusted total H2O/ACN ratio
calculated at the completion of the Fenton’s remediation. Vehicle controls were dosed
with a volume corresponding to the largest volume of reaction mixture tested in the

treatment dishes. Controls, which received no dose of chemical or Milli-Q H2O/ACN

142

solvent, were performed for each experiment as a means of evaluating a normal level of
GJIC and the overall “health” of the cells. By performing a t-test for each experiment, it
was found that the areas of dye spread for the control dishes did not vary significantly
from the areas for the vehicle controls at a 95% CI. Therefore, it could be concluded that
the 80/20 and 50/50 Milli-Q H2O/ACN solvents, at the volumes tested, were not a
significant source of inhibition in the experiments. Statistical analyses were performed
by means of the t-test and one-way analysis of variance (ANOVA) to compare the

chemical treatment results and vehicle control results.

6.3 Results and Discussion
6.3.1 Preliminary Methylene Blue Dye Tests

Figure 6.1 compares the MB dye test results from the investigations of 100%
ACN, 80/20 Milli-Q H2O/ACN solvent, and 50/50 Milli-Q H2O/ACN solvent with strips
tested with Milli-Q H20 and no sample (control). As shown in Figure 6.1A, the MB dye
test control strip (no sample added) was homogeneously dark blue in color. When the
MB dye test was performed with Milli-Q water (Figure 6.1B), no bleaching or
discoloration was observed. Similarly, when the MB dye test was performed with 100%
ACN (Figure 6.1C), no bleaching or discoloration was observed. Although neither Milli-
Q water nor 100% ACN alone resulted in bleaching or discoloration of the MB, Milli-Q
H2O/ACN solvent resulted in discoloration of the MB unique to the 80/20 and 50/50
volume ratios examined. When the MB dye test was performed with 80/20 Milli-Q
H2O/ACN solvent (Figure 6.1D), very slight diffuse discoloration of the MB to light blue

appeared with no dark blue outline. As shown in Figure 6.1E, a MB dye test of 50/50

143

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of Milli-Q H2O, (C) 40 (IL of 100% acetonitrile (ACN), (D) 40 uL of 80/20 Milli-Q
H2O/ACN, and (E) 40 uL of 50/50 Milli-Q H2O/ACN.

144

Milli-Q H2O/ACN solvent produced diffuse discoloration of the MB to light blue,
spreading out/emanating from the point of application, with a dark blue band surrounding
the discoloration. The results of the 80/20 Milli-Q H2O/ACN and 50/50 Milli-Q
H2O/ACN MB dye tests illustrate the importance of conducting MB dye tests of the
solvents to be used in performing experiments in which the presence of hydroxyl radicals
will be determined. Only by recognizing the appearance of the MB dye test results with
the solvents can the presence of hydroxyl radicals be identified in experiments.

Figure 6.2 compares the MB dye test results of 80/20 Milli-Q H2O/ACN solvent
adjusted to pH 3.6, 6.8, 8.3, and 10.0. For each pH tested, almost identical very slight
difiuse discoloration of the MB to light blue appeared with no dark blue outline. As was
observed for Milli-Q H2O with varying pH values (Chapter 4.3.5), the MB dye test

results for 80/20 Milli-Q H2O/ACN solvent are not influenced by sample pH.

6.3.2 Fenton’s Remediation of 80/20 and 50l50 Milli-Q Water/Acetonitrile

During the F enton’s remediation of 80/20 Milli-Q H2O/ACN solvent, prior to
quenching, no significant change in solution pH (~ 3.0) and temperature (22.5 °C)
occurred during the 60 minute reaction period. The unquenched Fenton’s reaction
mixture remained clear and colorless in appearance throughout the reaction period.
Figure 6.3 presents the MB dye test results for Fenton’s remediation of 80/20 Milli-Q
H2O/ACN solvent. The MB dye test control strip (no sample added) was homogeneously
dark blue in color (Figure 6.3A) and no bleaching or discoloration was observed for the
MB dye test performed with Milli-Q water (Figure 6.3B). When the MB dye test was

performed with 80/20 Milli-Q H2O/ACN solvent (Figure 6.3C), very slight diffuse

145

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blue dye tests were performed using 40 uL samples.

146

Figure 6.3 Fenton’s remediation of 80/20 Milli-Q H2O/ACN solvent with a Fe2+zH2O2
molar ratio of 1:20. Methylene blue dye test results for (A) control (no sample added);
(B) Milli-Q water; (C) 80/20 Milli-Q H2O/ACN; unquenched Fenton’s reaction mixture
at (D) 15 nrinutes, (E) 30 minutes, and (F) 60 minutes; and F enton’s reaction mixture
quenched with (G) 6 drops and (H) 11 drops of 10% Na2SO3. All methylene blue dye

tests were performed using 40 uL samples.

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discoloration of the MB to light blue appeared with no dark blue outline. As shown in
Figures 6.3D, E, and F, MB dye tests indicated the presence of hydroxyl radicals during
the reaction period (unquenched) by immediate concentrated light blue/almost white
discoloration (bleaching) with a dark blue outline at 15, 30, and 60 minutes, respectively.
Similar degrees of bleaching were observed at each of the reaction times. The very slight
diffuse discoloration observed for the MB dye test performed with 80/20 Milli-Q
H2O/ACN solvent might have occurred in the background of the unquenched MB dye
tests; however, the major discoloration can be attributed to the presence of hydroxyl
radicals due to the concentrated nature of the bleaching. Since hydroxyl radicals
appeared to be produced throughout the reaction period, the ACN does not appear to have
a scavenging effect on the hydroxyl radicals to the extent detectable by the MB dye test.
The bleaching observed for Fenton’s remediation of 80/20 Milli-Q H2O/ACN solvent
was similar in appearance to that observed for Fenton’s remediation in Milli-Q water
(Chapter 5).

The Fenton’s reaction was quenched after 60 minutes. The pH of the reaction
mixture increased from approximately 3.0 to 8.4. The temperature remained constant at
approximately 22.5 °C. The color of the reaction mixture changed from colorless to light
rust orange, attributable to the more basic pH of the reaction mixture during the
quenching process, which results in the conversion of iron from a hydrated ferrous form
to a colloidal ferric form and the formation of ferric hydroxide (8, 9, 10). To verify,
qualitatively, that quenching was complete, a MB dye test was performed. Additional
10% Na2S03 solution was added to the reaction mixture and the MB dye test was

repeated until the results resembled the MB dye test result for 80/20 Milli-Q H2O/ACN

149

solvent alone, indicating an absence of hydroxyl radicals. The MB dye test results of the
quenched F enton’s reaction mixtures are shown in Figures 6.3G and H. The very slight
white film, observed over the surface of the test area and most evident as a faint white
line at the edges of the test strip, can be attributed to excess 10% Na2SO3. Complete
quenching of the reaction was achieved by the addition of a total of 11 drops (0.42 mL)
of 10% Na2SO3 solution. As shown in Figure 6.3H, the MB dye test of the Fenton’s
reaction mixture, quenched with a total of 11 drops of 10% Na2SO3, produced results that
closely resemble the MB dye test result indicated for 80/20 Milli-Q H2O/ACN (Figure
6.3 C) and lack bleaching, thereby verifying that quenching was complete.

To aid in the removal of iron from the reaction mixture by precipitation of Fe“,
following quenching, the reaction mixture was adjusted to approximately pH 9.
Following this pH adjustment, the solution was a light rust orange color with very fine
light orange particles suspended in solution. Since the reaction mixture was not highly
concentrated with precipitate, centrifugation was not performed prior to filtration. The
reaction mixture was filtered and 2 mL of Milli-Q water was used for rinsing during the
filtration process. The filtrate appeared clear and colorless with no precipitate.
Following adjustment of the filtrate to pH 7, the solution remained clear and colorless
with no precipitate. The total H2O/ACN ratio (by volume) at the start of the remediation
experiment was 80/20 or 4.00. The adjusted total H2O/ACN ratio was calculated at the
completion of the experiment to be 4.99.

During the Fenton’s remediation of 50/50 Milli-Q H2O/ACN solvent, prior to
quenching, no significant change in solution pH (~ 3.0) and temperature (23.5 °C)

occurred during the 60 minute reaction period. The unquenched Fenton’s reaction

150

mixture remained clear and colorless in appearance throughout the reaction period.
Figure 6.4 presents the MB dye test results for Fenton’s remediation of 50/50 Milli-Q
H2O/ACN solvent. The MB dye test control strip (no sample added) was homogeneously
dark blue in color (Figure 6.4A) and no bleaching or discoloration was observed for the
MB dye test performed with Milli-Q water G’igure 6.4B). The MB dye test performed
with 50/50 Milli-Q H2O/ACN solvent (Figure 6.4C) produced diffuse discoloration of the
MB to light blue, spreading out/emanating fiom the point of application, with a dark blue
band surrounding the discoloration. As shown in Figures 6.4D, E, and F, MB dye test
strips tested with unquenched Fenton’s reaction mixture at 15, 30, and 60 minutes,
respectively, similarly indicated the presence of hydroxyl radicals by an immediate
concentrated white discoloration (bleaching) at the point of application surrounded by
diffuse discoloration of the MB to light blue, spreading out/emanating from the point of
application, with a dark blue band surrounding the discolored area. Since hydroxyl
radicals appeared to be produced throughout the reaction period, the ACN does not
appear to have a scavenging effect on the hydroxyl radicals to the extent detectable by the
MB dye test.

The diffuse discoloration surrounding the area of bleaching closely resembled the
diffirse discoloration observed for the MB dye test of 50/50 Milli-Q H2O/ACN solvent
(Figure 6.4C), and might be explained by the effect of 50/50 Milli-Q H2O/ACN solvent
occurring in the background of the hydroxyl radical bleaching of the MB. Unlike the
unquenched Fenton’s reaction mixture for 80/20 Milli-Q H2O/ACN, an area of diffuse
discoloration for the unquenched Fenton’s reaction mixture for 50/50 Milli-Q H2O/ACN

appeared to spread beyond the area of bleaching. Since the diffuse discoloration of the

151

Figure 6.4 Fenton’s remediation of 50/50 Milli-Q H2O/ACN solvent with a Fe2+zH2O2
molar ratio of 1:20. Methylene blue dye test results for (A) control (no sample added);
(B) Milli-Q water; (C) 50/50 Milli-Q H2O/ACN; unquenched Fenton’s reaction mixture
at (D) 15 minutes, (E) 30 minutes, and (F) 60 minutes; and F enton’s reaction mixture
quenched with (G) 6 drops and (H) 8 drops of 10% Na2SO3. All methylene blue dye tests

were performed using 40 uL samples.

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80/20 Milli—Q H2O/ACN solvent (Figure 6.3C) was less pronounced than that observed
for 50/50 Milli-Q H2O/ACN solvent (Figure 6.4C), the background effect of the 80/20
Milli-Q H2O/ACN solvent might have been concealed by the bleaching that occurred for
the unquenched Fenton’s reaction mixture for 80/20 Milli-Q H2O/ACN. Furthermore,
the dark blue band surrounding the discolored area of the MB dye test performed with the
unquenched Fenton’s reaction mixture for 50/50 Milli-Q H2O/ACN closely resembled the
dark blue band observed for the MB dye test of 50/50 Milli-Q H2O/ACN solvent (Figure
6.4C). In addition, the dark blue band was dissimilar from the dark blue outline observed
for the unquenched Fenton’s reaction mixture for 80/20 Milli-Q H2O/ACN, which closely
followed the contour of the area of bleaching. The results of the unquenched Fenton’s
reaction mixtures for 80/20 Milli-Q H2O/ACN and 50/50 Milli-Q H2O/ACN MB dye
tests further emphasize the aforementioned importance of conducting MB dye tests of the
solvents to be used in performing experiments in which the presence of hydroxyl radicals
will be determined.

The F enton’s reaction was quenched after 60 minutes. The pH of the reaction
mixture increased from approximately 3.0 to 8.8. No significant change occurred in the
temperature of the reaction mixture. Following quenching, very few fine rust colored
particles appeared in the reaction mixture, attributable to the more basic pH of the
reaction mixture during the quenching process, which results in conversion of iron fi'om a
hydrated ferrous form to a colloidal ferric form and the formation of ferric hydroxide (8,
9, 10). To verify, qualitatively, that quenching was complete, a MB dye test was
performed. Additional 10% Na2S03 solution was added to the reaction mixture and the

MB dye test was repeated until the results resembled the MB dye test result for the 50/50

154

Milli-Q H2O/ACN solvent alone, indicating an absence of hydroxyl radicals. The MB
dye test results of the quenched F enton’s reaction mixtures are shown in Figures 6.4G
and H. Complete quenching of the reaction was achieved by the addition of a total of 8
drops (0.36 mL) of 10% Na2SO3 solution. As shown in Figure 6.4H, the MB dye test of
the F enton’s reaction mixture, quenched with a total of 8 drops of 10% Na2SO3, produced
results that closely resemble the MB dye test result indicated for 50/50 Milli-Q H2O/ACN
(Figure 6.4C) and lack concentrated white discoloration (bleaching), thereby verifying
that quenching was complete.

To aid in the removal of iron fiom the reaction mixture by the precipitation of
F e3 +, following quenching, the reaction mixture was adjusted to approximately pH 9.
Following this pH adjustment, the solution was clear with very few fine rust colored
particles in suspension. Since the reaction mixture was not highly concentrated with
precipitate, no centrifugation was performed prior to filtration. The reaction mixture was
filtered and 1 mL of Milli-Q water was used for rinsing during the filtration process. The
filtrate appeared clear andcolorless with no precipitate. Following adjustment of the
filtrate to pH 7, the solution remained clear and colorless with no precipitate. The total
H2O/ACN ratio (by volume) at the start of the remediation experiment was 50/50 or 1.00.
The adjusted total H2O/ACN ratio was calculated at the completion of the experiment to

be 1.22.
6.3.3 In Vitro Bioassay for GJIC

Prior to and following warming to room temperature, the final reaction mixture

solutions from Fenton’s remediation of 80/20 Milli-Q H2O/ACN and 50/50 Milli-Q

155

H2O/ACN solvents were clear and colorless with no precipitate. Figure 6.5 presents the
30 minute incubation dose-response GJIC bioassay results for a volume range of 0 to 150
uL of final reaction mixture solution from the Fenton’s remediation of 80/20 Milli-Q
H2O/ACN solvent. Figure 6.6 presents the 30 minute incubation dose-response GJIC
bioassay results for a volume range of 0 to 60 uL of final reaction mixture solution from
Fenton’s remediation of 50/50 Milli-Q H2O/ACN solvent. For each of the final reaction
mixture solutions from Fenton’s remediation of 80/20 Milli-Q H2O/ACN and 50/50
Milli-Q H2O/ACN solvents, no inhibition of GJIC was observed with 30 minutes of
incubation time. For the highest tested volume (150 uL) of the final reaction mixture
solution from the F enton’s remediation of 80/20 Milli-Q H2O/ACN solvent, no inhibition
of GJIC was observed at 30 minutes (FOC = 1.02 i 0.03) and 2 hours (FOC = 1.01 i
0.02) of incubation. Likewise, for the highest tested volume (60 uL) of final reaction
mixture solution from Fenton’s remediation of 50/50 Milli-Q H2O/ACN solvent, no
inhibition of GJIC was observed at 30 minutes (F OC = 1.01 i 0.03) and 2 hours (FOC =
1.02 :l: 0.02) of incubation.

Since no inhibition of GJIC was observed for the dose-response GJIC bioassay
results for each of the final reaction mixture solutions fiom the Fenton’s remediation of
80/20 Milli-Q H2O/ACN and 50/50 Milli-Q H2O/ACN solvents for incubation times of
30 minutes and 2 hours, it was decided that cytotoxicity, time-response, and time of
recovery bioassays were not required. In addition, since the final reaction mixture
solutions produced from a 60 minute F enton’s reagent reaction did not produce any
inhibition of GJIC in the dose-response bioassays, it was decided that the investigation of

longer reaction times (5 hours and 24 hours) were also unnecessary. Although some

156

 

 

 

1°0ka N 4 ‘1

0.75 - ..

GJIC
(Fraction of the Control)

0.50 - . ..

 

 

 

0.25 )- d
0.00 L I I I I I L I I
0 20 4O 60 80 100 120 140 160
Volume of Sample Added (IIL)

Figure 6.5 Dose-response GJIC bioassay results with a 30 rrrinute incubation time for a
volume range of 0 to 150 uL of a solution resulting from Fenton’s reagent remediation
with only 80/20 Milli-Q water/ACN solvent (no PCB), a Fe2+zH2O2 ratio of 1:20, and 60
minutes reaction time. Each data point is representative of the results for a set of
chemically treated triplicates reported as an average F OC i standard deviation
determined at the 95% confidence interval.

157

 

 

 

 

I I I I I I I
1.00 L W 1
i
o 0.75 L- ..
O
2%
3‘s
5 0.50 - -
3;
0
E
E:
0.25 b q
000 I 4 l 1 l L l
o 10 20 30 4o 50 so
Volume of Sample Added (uL)

Figure 6.6 Dose-response GJIC bioassay results with a 30 minute incubation time for a
volume range of 0 to 60 uL of a solution resulting from Fenton’s reagent remediation
with only 50/50 Milli-Q water/ACN solvent (no PCB), a Fe2+zH2O2 ratio of 1:20, and 60
minutes reaction time. Each data point is representative of the results for a set of
chemically treated triplicates reported as an average FOC i standard deviation
determined at the 95 % confidence interval.

158

researchers have used reaction times of up to 1 week (11, 12), a 60 minute reaction time

is consistent with that used by Trapido et al. (4).

6.4 Conclusions

F enton’s remediation of the solvents 80/20 Milli-Q H2O/ACN and 50/50 Milli-Q
H2O/ACN (by volume) were investigated to determine the toxicological effect of ACN in
combination with water as a solvent. In addition, the use of the MB dye test for Fenton’s
reaction in Milli-Q H2O/ACN was examined. Although neither Milli-Q water nor 100%
ACN alone resulted in bleaching or discoloration of the MB, Milli-Q H2O/ACN solvent
resulted in the discoloration of the MB unique to the 80/20 and 50/50 volume ratios
examined. The results of the 80/20 Milli-Q H2O/ACN and 50/50 Milli-Q H2O/ACN MB
dye tests illustrated the importance of conducting MB dye tests of the solvents to be used
in performing experiments, in which the presence of hydroxyl radicals will be
determined. Only by recognizing the appearance of the MB dye test results with the
solvents can the presence of hydroxyl radicals be identified in these experiments. As was
observed for Milli-Q H2O with varying pH values (Chapter 4.3.5), the MB dye test
results for 80/20 Milli-Q H2O/ACN solvent were not influenced by the sample pH. The
presence of hydroxyl radicals in a reaction mixture was indicated in the MB dye test by
an immediate concentrated white discoloration (bleaching) characteristic to the solvent
being used. MB dye tests indicating the absence of hydroxyl radicals in a reaction
mixture were identified by a resemblance to the MB dye test results for the respective
Milli-Q H2O/ACN solvent alone and a lack of bleaching. In the MB dye tests of

unquenched Fenton’s reaction mixtures, depending on the solvent, the discoloration

159

effect of the solvent was observed to occur in the background of the hydroxyl radical
bleaching of the MB. Since hydroxyl radicals appeared to be produced throughout the
reaction period, the ACN does not appear to have a scavenging effect on the hydroxyl
radicals to the extent detectable by the MB dye test.

For each of the final reaction mixture solutions from the Fenton’s remediation of
80/20 Milli-Q H2O/ACN and 50/50 Milli-Q H2O/ACN solvents, no inhibition of GJIC
was observed with 30 minutes of incubation time for volume ranges of 0 to 150 uL and 0
to 60 uL, respectively. Furthermore, for the highest tested volumes of the final reaction
mixture solutions, no inhibition of GJIC was observed at 2 hours of incubation. Hence,
Fenton’s remediation of 80/20 Milli-Q H2O/ACN and 50/ 50 Milli-Q H2O/ACN solvents
alone cannot be expected to result in any toxicity, and any toxicity resulting from the
Fenton’s remediation of 4,4'DCBP in either of these solvents can be assumed to be
independent of the influence of these solvents. The remediation procedure further
developed in this series of experiments was later applied to the Fenton’s remediation of

4,4'DCBP.

160

6.5 References

l.

10.

11.

12.

Micaroni, R.C.C.M.; Bueno, M.I.M.S.; Jardim, W.F. Degradation of Acetonitrile
Residues Using Oxidation Processes. J. Braz. Chem. Soc. 2004, 15 (4), 509-513.

Satoh, A.Y.; Trosko, J.E.; Masten, S.J. Epigenetic Toxicity of Hydroxylated
Biphenyls and Hydroxylated Polychlorinated Biphenyls on Normal Rat Liver
Epithelial Cells. Environ. Sci. Technol. 2003, 3 7 (12), 2727-2733.

Satoh, A.Y.; Trosko, J .E.; Masten, S.J. Methylene Blue Dye Test for Rapid
Qualitative Detection of Hydroxyl Radicals Formed in a Fenton’s Reaction Aqueous
Solution. Environ. Sci. T echnol. 2007, 41 (8), 2881-2887.

Trapido, M.; Goi, A. Degradation of nitrophenols with the Fenton reagent. Proc.
Estonian Acad Sci. Chem. 1999, 48(4), 163-173.

Dutta, K.; Mukhopadhyay, S.; Bhattacharjee, S.; Chaudhuri, B. Chemical oxidation of
methylene blue using a F enton-like reaction. J. Hazard Mater. 2001, 384(1), 57-71.

Mohamadin, A.M. Possible role of hydroxyl radicals in the oxidation of
dichloroacetonitrile by Fenton-like reaction. J. Inorg. Biochem. 2001, 84 (1-2), 97-
105.

Trapido, M. Tallinn Technical University, Tallinn, Estonia. Personal Communication,
2002.

Lindsey, M.E.; Tarr, M.A. Quantitation of hydroxyl radical during Fenton oxidation
following a single addition of iron and peroxide. Chemosphere 2000, 41 (3), 409-417.

Arnold, S.M.; Hickey, W.J.; Harris, R.F. Degradation of atrazine by Fenton’s
reagent: condition optimization and product quantification. Environ. Sci. T echnol.
1995, 29(8), 2083-2089.

Pratap, K.; Lemley, A.T. Fenton electrochemical treatment of aqueous atrazine and
metolachlor. J. Agric. Food Chem. 1998, 46 (8), 3285-3291.

Sedlak, D.L.; Andren, A.W. Aqueous-Phase Oxidation of Polychlorinated Biphenyls
by Hydroxyl Radicals. Environ. Sci. T echnol. 1991, 25(8), 1419-1427.

Dercova, K.; Branislav, V.; Tandlich, R.; Subova, L. Fenton's Type Reaction and
Chemical Pretreatment of PCBs. Chemosphere 1999, 39(15), 2621-2628.

161

Chapter 7

Fenton’s Remediation of 4,4'-Dichlorobiphenyl in 50/50 Milli-Q Water/Acetonitrile
and Toxicity of Remediation Mixture

7.1 Introduction

Current remediation practices often emphasize the disappearance of the parent
compound to at or below regulatory limits, but often disregard the importance of reducing
the overall toxicity. Since remediation byproducts can exhibit equal or greater toxicity
than the parent compound, it is important to consider in a remediation process not only
the removal of the parent compound, but also the toxicological impact of the remediation
byproducts (1). Although the reaction of F enton’s reagent with various PCBs has been
investigated (2, 3), the subsequent toxicity and characterization of remediation
byproducts have not been extensively studied. This chapter investigates the toxicological
analyses of the solution resulting from Fenton’s remediation of 4,4'-dichlorobiphenyl
(4,4’DCBP). The disappearance of 4,4'-dichlorobiphenyl, as a result of Fenton’s
remediation, is presented in Chapter 8.

A combination of Milli-Q H20 and acetonitrile was selected as the solvent for
dissolving 4,4'DCBP in the Fenton’s remediation experiments. Acetonitrile (ACN) was
added as part of the solvent to increase the solubility of the 4,4'DCBP. It has been shown
that the presence of the acetonitrile does not inhibit the Fenton’s reaction, as it is not
appreciably reactive with hydroxyl radicals (4), and, in toxicology studies, it does not
affect gap junctional intercellular communication (GJIC) at low concentrations (5). In
Chapter 6, F enton’s remediation of the solvents 80/20 Milli-Q H2O/ACN and 50/50

Milli-Q H2O/ACN (by volume) were investigated to determine the toxicological effect of

162

ACN in combination with water as a solvent in remediation. Since neither solvent
resulted in the inhibition of GJIC for the volume ranges and incubation times tested, any
toxicity resulting from the F enton’s remediation of 4,4'DCBP in either of these solvents
can be assumed to be independent of the influence of these solvents.

Fenton’s remediation of a solution of 4,4'DCBP in 80/20 Milli-Q H2O/ACN was
performed for 60 minutes (with reaction conditions pH 3.0, temperature 23.0 °C,
Fe2+zH2O2 molar ratio 1:20, initial Fe2+ concentration 0.15 mM, and initial H202
concentration 3 mM); however, 80/20 Milli-Q H2O/ACN was determined to be an
inappropriate solvent for 4,4'DCBP because of the insolubility of 4,4'DCBP. Throughout
the remediation process, small white particles were observed in the reaction mixture and
white residue adhered to the surface of the centrifuge tube and thermometer. No decrease
in particles or residue occurred as the reaction progressed. Although the final Fenton’s
remediation solution was clear and colorless, since the white particles were removed by
filtration and the white residue remained on the surface of the centrifuge tube and
thermometer, toxic chemicals generated during the reaction might have been lost. For
Fenton’s remediation of 4,4'-dichlorobiphenyl, 50/50 Milli-Q H2O/ACN was therefore

selected as the solvent.

7.2 Experimental Section
7.2.1 Chemicals

For the Fenton’s remediation portion of this section of research the following
chemicals were used. 4,4'-Dichlorobiphenyl (99% purity) was purchased from Chem

Service Inc. (West Chester, PA). Acetonitrile (99.8% purity) was purchased from EM

163

Science (Gibbstown, NJ). Methylene blue dye (3,7-bis(dimethylamino)-phenothiazin-5-
ium chloride) (97% purity) was purchased from Fluka (Buchs, Switzerland). Thirty-
percent hydrogen peroxide (H202) (unstabilized), iron(II) sulfate heptahydrate

(F eSO4-7H2O) (99% purity), and sodium sulfite (Na2SO3) (anhydrous, 98% purity) were
purchased from Sigma-Aldrich (St. Louis, MO). Sulfuric acid (H2SO4) (96% purity) and
sodium hydroxide (NaOH) pellets (99% purity) were purchased from J .T. Baker
(Phillipsburg, NJ). Throughout this chapter and dissertation, Milli-Q water was obtained
fi'om a Milli-Q Ultrapure Water Purification System (System Type ZMQS6VFOY)
purchased from Millipore Corp. (Bedford, MA).

For the toxicology portion of this section of research the following chemicals
were used. For cell culture, D-medium (Formula No. 78-5470 EG), Fetal Bovine Serum
(F BS), and Gentamicin were purchased fi'om GIBCO Laboratories (Grand Island, NY).
Lucifer yellow CH, dilithium salt, was purchased from Sigma Chemical Co. (St. Louis,
MO). Formaldehyde solution (3 7%) for the GJIC bioassays was purchased from J .T.

Baker (Phillipsburg, NJ).

7.2.2 Methods

7.2.2.1 Fenton’s Remediation of 4,4'-Dichlorobiphenyl in 50150 Milli-Q H2O/ACN
Fenton’s remediation of a solution of 4,4'DCBP in 50/50 Milli-Q H2O/ACN (by

volume) was performed for 60 minutes. The initiation of remediation was defined as the

moment 3% H202 was added. Based on the results of Fenton’s remediation in Milli-Q

water (Chapter 5), an Fe2+:H2O2 molar ratio of 1:20 was used for the Fenton’s reaction.

The initial concentrations of Fe2+ and H202 in the reaction rrrixture were 0.15 mM and 3

164

mM, respectively, which are within the ranges previously used by Trapido et al. (6). The
volume of 5.0 mM FeSO4 and 3% H202 added to the reaction mixture was calculated to
reflect the assumption that this addition caused a significant change in the overall
reaction mixture volume. The amount of water added by addition of 5 .0 mM F eSO4, pH
adjustment, addition of 3% H202, quenching, and rinsing of the filtration funnel was
monitored throughout the experiment, and an adjusted total H2O/ACN ratio was
calculated at the completion of the experiment. In this calculation, the volumes of 5.0
mM FeSO4, 3% H202, 10% Na2SOg, 0.5 M H2SO4, and 1M NaOH added were assumed
to be 100% water. The total H2O/ACN ratio suggested the degree of dilution of the
sample that occurred during the experiment. In each experiment, this ratio was
maintained as constant as possible to allow for comparison of the results. The pH and
temperature were monitored throughout the experiments.

Methylene blue (MB) dye tests were performed during the Fenton’s reaction to
verify the formation of hydroxyl radicals and during the quenching process to verify the
completion of quenching (absence of hydroxyl radicals). MB dye test strips were
prepared by the method of Satoh et al. (7) as described in detail in Chapter 4. Forty
microliters of liquid sample were placed dropwise onto the center of the MB dyed section
of a test strip, allowing for absorption between drops. The MB dye tests were compared
against the test strip tested with 50/50 Milli-Q H2O/ACN solvent, which is characterized
by a diffuse discoloration of the MB, spreading out/emanating from the point of
application, with a dark blue band surrounding the area of discoloration. MB dye tests
indicating the absence of hydroxyl radicals in a reaction mixture with a 50/ 50 Milli-Q

H2O/ACN solvent were identified by a resemblance to the MB dye test results for 50/50

165

Milli-Q H2O/ACN solvent alone. Discoloration of the MB dye, due to the presence of
hydroxyl radicals in a reaction mixture with a 50/ 50 Milli-Q H2O/ACN solvent, was
indicated by an immediate concentrated white discoloration (bleaching) at the point of
application surrounded by diffuse discoloration of the MB to light blue, spreading
out/emanating from the point of application, with a dark blue band surrounding the
discolored area.

This experiment involved the remediation of 8.2 mg of 4,4’DCBP. A 50 mL
centrifuge tube with a vaned tube stir bar was used as the reaction vessel. The 4,4'DCBP
was dissolved in 50/ 50 Milli-Q H2O/ACN (by volume), in a step-wise procedure, to
obtain a final concentration of 2 mM 4,4'DCBP (the final concentration of the test
solution used in the 4,4'DCBP toxicology study, Chapter 3). To improve the dissolution
of 4,4'DCBP, it was first dissolved in the ACN portion of the 50/50 Milli-Q H2O/ACN
solvent, and then the Milli—Q H2O portion was added in 1 mL increments. The reaction
mixture was sonicated following each addition of the solvent. A 5.0 mM FeSO4 stock
solution was prepared from FeSO4-7H2O and Milli-Q water. The 5.0 mM FeSO4 stock
solution was then added to the reaction mixture at the calculated volume to achieve a
final concentration of 0. 1 5 mM F e“. For optimal Fenton’s reaction efficiency, the
reaction mixture was adjusted to pH 3 with 0.5 M H2SO4 and/or 1 M NaOH. The pH was
monitored using a 720A plus pH/ISE meter with an 8102 BNU Ross Ultra Combination
pH electrode (ThermoOrion, Beverly, MA).

Following the adjustment of the reaction mixture to pH 3, the F enton’s reaction
was initiated by the addition of 3% H202, prepared from 30% unstabilized H202 and

Milli-Q water, to the reaction mixture to obtain an initial concentration of 3 mM H202.

166

Since stabilizing agents (hydroxyl radical scavengers) in commercial H202 might affect
the results (8), only H202 devoid of stabilizing agents was used. In order to prevent
localized reactions (that might occur when a small volume of very concentrated solution
is added to a reaction mixture), 3% H202 rather than 30% H2O2 was used to initiate the
reaction. The remediation reaction was allowed to occur for 60 minutes with occasional
sonication of the reaction mixture. To test, qualitatively, the production of hydroxyl
radicals during the reaction, MB dye tests were performed on the unquenched reaction
mixture at varying times during the remediation. In addition, MB dye tests were
performed on 40 11L samples of Milli-Q water and 50/50 Milli-Q H2O/ACN solvent.
After the 60 minute remediation reaction period, the F enton’s reaction was
quenched with a 10% Na2303 solution (w/v), prepared from Na2803 and Milli-Q water.
As recommended by Trapido et al. (6, 9), the reaction was quenched by the addition of 2
to 3 drops of 10% Na2SO3 solution for every 10 mL of reaction mixture. A 0.5 mL glass
pipette was used to administer the drops to the reaction mixture. To verify, qualitatively,
that quenching was complete, 5 minutes after the addition of the 10% Na2803 solution, a
MB dye test was performed. An additional aliquot of 10% Na2803 solution was added to
the reaction mixture, and the MB dye test was repeated until an absence of hydroxyl
radicals in the reaction mixture was indicated (quenching was complete). To aid in the
removal of iron from the reaction mixture by precipitation of F e3 +, following quenching,
the reaction mixture was adjusted to pH 9 with 0.5 M H2SO4 and/or 1 M NaOH. If a
large amount of precipitation in the reaction mixture was observed, the solution was
centrifuged at 1200 rpm for 10 minutes and the supernatant was retained for filtration.

The reaction mixture or supernatant was then filtered through a 1.0 pm glass fiber filter,

167

using a vacuum filtration unit, into a large culture tube inserted into a 1000 mL
Erlenmeyer side-arm filtration flask. Milli-Q water was used for rinsing during filtration
only if the reaction mixture touched the sides of the fimnel or there was residue in the
centrifuge tube. The filtered reaction mixture was transferred to a clean 50 mL centrifuge
tube containing a vaned tube stir bar. The filtered solution was then adjusted to pH 7
with 0.5 M H2SO4 and/or 1 M NaOH to neutralize the reaction mixture pH for the cells in
the GJIC bioassays. This final Fenton’s remediation solution was transferred to an
amber, 60 mL Bosron round bottle with a TFE closure and was refrigerated at 4 °C until

toxicology bioassays were performed.

7.2.2.2 Cell Culture Techniques

WB—F344 rat liver epithelial cells were obtained from Dr. J. W. Grisham and Dr.
M. S. Tsao of the University of North Carolina (Chapel Hill, NC) (I). This cell line was
selected because it is a diploid, nontumorigenic cell line originating from a strain of rat
that has been used for toxicological/cancer studies of numerous chemicals, thereby
allowing for a source of comparison (1 ). Since 70% of the chemicals that are carcinogens
are liver carcinogens and the liver is the “first pass” organ for ingested toxins, liver cells
are important for toxicological/cancer studies (10). Furthermore, the WB-F344 cell line
was designed for in vitro assays to match the many in vivo tumor promotion assays that
had been done in rat liver, specifically, in the Fischer 344 rat.

The cell culture techniques performed were similar to those described by Hemer
et al. (I) and Luster-Teasley et al. (11). Cells were cultured in 150 cm2 sterile, treated,

polystyrene cell culture flasks (Corning Inc., Corning, NY) in 25 mL of D-mediurn

168

containing 5% Fetal Bovine Serum (FBS) and 0.2% Gentamicin. The cells were
incubated at 37 °C in a water-j acketed IR Autoflow Automatic CO2 incubator (NUAIRE,
Inc., Plymouth, MN) in a humidified atmosphere with 5% CO2 and 95% air. The time
required for cell growth confluency was about two days. The confluent culture was split
and transferred every other day into a new 150 cm2 culture flask with new medium
mixture. In addition, from 150 cm2 flasks of confluent cells, cultures were prepared for
the bioassays in 35 mm diameter, sterile, treated polystyrene cell culture dishes (Corning
Inc., Corning, NY) with 2 mL of D-medium supplemented with 5% FBS. The cultures

for the bioassays were incubated under the same conditions as the aforementioned flasks.

7.2.2.3 In Vitro Bioassay for GJIC

The GJIC bioassays were performed on confluent WB-F344 cell cultures (usually
2 days of growth) grown in 35 mm diameter culture dishes as described in the preceding
section. The scrape-loading/dye transfer (SL/DT) procedure for determining the GJIC
was adapted from the method described by El-Fouly et al. (12) and is described in detail
by Hemer et al. (1 ). A detailed description of the spread of Lucifer yellow dye from the
scrape to neighboring cells can be found in Wilson et al. (13). Chemical treatments (final
F enton’s remediation solution), controls (no dose), and vehicle controls (50/50 Milli-Q
H2O/ACN solvent) were performed in triplicates. All GJIC bioassay results assumed that
the filtration process performed during the Fenton’s remediation experiment removed no
toxic chemicals generated during the reaction. Since the exact molecular weight and
concentration of the reaction mixture solution were unknown, GJIC bioassay doses were

investigated in terms of volumes rather than concentrations. Since slight inhibition of

169

GJIC occurs at a final ACN concentration of 2.0% in the cell culture medium
(corresponding to 40 uL of ACN), all GJIC bioassays were conducted with a final ACN

concentration of 1.5% or less (corresponding to 30 pL of ACN or less) (5). Therefore,
the maximum dose volume that could be tested for the resulting solution from Fenton’s
remediation of 4,4'DCBP in 50/50 Milli-Q H2O/ACN was 60 uL.

Doses were applied directly to the dishes of confluent cell cultures. Vehicle
controls for the GJIC bioassays were performed using 50/50 Milli-Q H2O/ACN solvent
and were incubated for the same incubation times as chemical treatments. To be
conservative, the 50/ 50 Milli-Q H2O/ACN solvent was used for the vehicle controls
instead of a solution based on the adjusted total H2O/ACN ratio calculated at the
completion of the Fenton’s remediation. Vehicle controls were dosed with a volume
corresponding to the largest volume of final Fenton’s remediation solution tested in the
treatment dishes. Controls, which received no dose of final Fenton’s remediation
solution or 50/50 Milli-Q H2O/ACN solvent, were performed for each experiment as a
means of evaluating a normal level of GJIC and the overall “health” of the cells.

For reasons to be discussed later in this chapter, based on results which indicated
that further toxicology studies were unnecessary, only dose-response and time-response
GJIC bioassays were performed. The volumes tested in the dose-response GJIC bioassay
for the final Fenton’s remediation solution were 0, 10, 20, 30, 40, SO, and 60 uL.
Confluent cells were exposed to the test volumes and allowed to incubate for 30 minutes
before assaying for GJIC. The time-response GJIC bioassay was used to determine the
effect of chemical exposure time on intercellular communication. For the time-response

experiments, confluent cultures of cells were exposed to a fixed volume of final Fenton’s

170

remediation solution for varying amounts of time ranging from 0 to 24 hours (1440
minutes) followed by GJIC bioassays. The test volume selected for the time-response
experiment was selected as the highest volume that causes a substantial amount of
inhibition at 30 minutes of exposure.

All culture dishes were examined within 24 hours of completion of the
experiment. Each culture dish of cells was digitally photographed such that the observed
scrape spanned the full horizontal width of the picture. A COHU High Performance
Color CCD Camera (Cohu, Inc., San Diego, CA) with a magnification of 200x under a
Nikon TE300 Eclipse Inverted Microscope (Nikon Corp., Japan) with a Nikon HB-
10103AF Super High Pressure Mercury 100W lamp (Nikon Corp., Japan) was used.

The fluorescence of the Lucifer yellow dye was used to determine the distance the
dye traveled perpendicular to the scrape. This distance of dye travel was indicative of the
level of GJIC within the culture. Quantitative analysis of the distance of dye spread was
performed using NucleoTech GelExpert software (NucleoTech Corp., Hayward, CA).
The distance of dye spread was measured in terms of the area of dye spread, by tracing
manually via free object quantification the area of farthest visible fluorescence. Since the
width of the photographed section was the same for every culture dish, measuring the
area of the dye spread was equivalent to measuring the distance of dye spread
perpendicular to the scrape. The area of dye spread for each chemical treatment (final
Fenton’s remediation solution) dish was compared to a control group of cells that were
exposed to 50/50 Milli-Q H2O/ACN solvent only (vehicle controls) under the same assay
as the treated cells. For each chemically treated dish, the fraction of the control was

calculated as the area of dye spread in the treated dish divided by the average area of dye

171

spread in the triplicate set of vehicle control dishes. The results for each set of
chemically treated triplicates were reported as an average fiaction of the control (F OC) :1:
standard deviation (SD) determined at the 95% confidence interval (CI).

The level of GJIC in cells exposed to the chemical treatment was assessed by the
decrease in communication of the cells as compared to the vehicle control groups. A
decrease in FOC corresponds directly to a decrease in GJIC (where the doses are not
cytotoxic). Interpretations of GJIC results are consistent with Luster-Teasley et al. (11)
and Hemer et al. (1). Complete communication (100%) between the cells is identified as
a FOC value of 1.0 as seen in the vehicle control. A FOC value greater than 0.9 is
difficult to statistically distinguish from the vehicle controls. FOC values between 0.9
and 0.5 indicate partial inhibition of GJIC. A F OC value less than or equal to 0.5 is
indicative of a significant amount of inhibition of GJIC, since this would be
representative of communication levels that are 50% or less than the normal
communication levels. F OC values between 0.3 and 0.0 are representative of complete
inhibition of GJIC. A F OC value of 0.3 is usually used to represent complete inhibition,
~ as it corresponds to the width of a single row of cells with no dye spreading beyond its
boundaries (1). By performing a t-test for each experiment, it was found that the areas of
dye spread for the control dishes did not vary significantly from the areas for the vehicle
controls at a 95% CI. Therefore, it could be concluded that the 50/50 Milli-Q H2O/ACN
solvent, at the volume tested, was not a significant source of inhibition in the
experiments. Statistical analyses were performed by means of the t-test and One Way
Analysis of Variance (AN OVA) to compare the chemical treatment results and vehicle

control results.

172

7.3 Results and Discussion
7.3.1 Fenton’s Remediation of 4,4'-Dichlorobiphenyl in 50/50 Milli-Q H20/ACN

Fenton’s remediation of a solution of 4,4'DCBP in 50/50 Milli-Q H2O/ACN (by
volume) was performed for 60 minutes. To improve the dissolution of 4,4'DCBP, it was
dissolved in 50/50 Milli-Q H2O/ACN in a step-wise procedure to obtain the final
concentration of 2 mM 4,4'DCBP. When the 4,4'DCBP was first dissolved in the ACN
portion of the 50/50 Milli-Q H2O/ACN solvent, the resulting solution was clear and
colorless with no precipitate or oily residue. The solution appearance remained
unchanged during the addition of the Milli-Q H2O portion of the 50/50 Milli—Q
H2O/ACN solvent until 5.19 mL of Milli-Q H2O had been added. Further addition of
Milli-Q H2O resulted in a light milky white solution. As the remaining Milli-Q H2O
portion was added in 1 mL increments, increasing the length of time the sample was
sonicated was necessary in order to ensure complete dissolution. Following the addition
of 5.0 mM FeSO4, pH adjustment, and sonication, the reaction mixture was clear and
colorless with very fine white particles in suspension. For the F enton’s remediation of
50/50 Milli-Q H2O/ACN solvent (discussed in Chapter 6), the reaction mixture remained
clear and colorless with no precipitate or milky appearance. Therefore, the light milky
white appearance and very fine white particles observed for Fenton’s remediation of a
solution of 4,4'DCBP in 50/50 Milli-Q H2O/ACN can be attributed to the presence of
4,4'DCBP in the solvent and is not a property of the solvent itself.

Following the adjustment of the reaction mixture to pH 3, the Fenton’s reaction
was initiated by the addition of 3% H202. The remediation reaction was allowed to occur

for 60 minutes with occasional sonication of the reaction mixture. During the Fenton’s

173

reaction remediation period, no significant change occurred in the solution pH (~ 3.0) and
temperature (23.0 °C). The solution remained clear and colorless with the amount of
very fine white particles in suspension decreasing as the reaction progressed, until no
precipitate was apparent, after about 5 minutes. No white residue adhered to the surface
of the centrifuge tube and thermometer.

In addition to performing MB dye tests on 40 uL samples of Milli-Q water and
50/50 Milli-Q H2O/ACN, MB dye tests were also performed on 40 uL samples of the
unquenched reaction mixture to verify the production of hydroxyl radicals during the
reaction. Figure 7.1 presents the MB dye test results for Fenton’s remediation of
4,4'DCBP in 50/50 Milli-Q H2O/ACN. As shown in Figure 7.1A, the MB dye test
control strip (no sample added) was homogeneously dark blue in color. When the MB
dye test was performed with Milli-Q water (Figure 7.1B), no bleaching or discoloration
was observed. As shown in Figure 7.1C, a MB dye test of 50/50 Milli-Q H2O/ACN
solvent produced diffuse discoloration of the MB to light blue, spreading out/emanating
from the point of application, with a dark blue band surrounding the discoloration. As
shown in Figures 7. 1 D, E, and F, MB dye test strips tested with unquenched Fenton’s
reaction mixture at 15, 30, and 60 minutes, respectively, similarly indicated the presence
of hydroxyl radicals by an immediate concentrated white discoloration (bleaching) at the
point of application surrounded by diffuse discoloration of the MB to light blue,
spreading out/emanating from the point of application, with a dark blue band surrounding
the discolored area. The diffuse discoloration surrounding the area of bleaching closely
resembled the diffuse discoloration observed for the MB dye test of 50/50 Milli-Q

H2O/ACN solvent (Figure 7.1C), and might be explained by the effect of 50/50 Milli-Q

174

Figure 7.1 Fenton’s remediation of a solution of 4,4'-dichlorobiphenyl in 50/50 Milli-Q
H2O/ACN. Methylene blue dye test results for (A) control (no sample added); (B) Milli-
Q water; (C) 50/50 Milli-Q H2O/ACN solvent; unquenched Fenton’s reaction mixture at
(D) 15 minutes, (E) 30 minutes, and (F) 60 minutes; and Fenton’s reaction mixture
quenched with (G) 6 drops and (H) 8 drops of 10% Na2SO3. All methylene blue dye tests
were performed using 40 uL samples. The Fenton’s reaction conditions were pH 3.0,
temperature 23.0 °C, F e2+zH2O2 molar ratio 1:20, initial Fe2+ concentration 0.15 mM, and
initial H202 concentration 3 mM. After a 60 minute remediation reaction period, the

F enton’s reaction was quenched with a 10% Na2803 solution (w/v). Following
quenching with 10% Na2SOg, the F enton’s reaction mixture pH increased to 9.0 and the

temperature remained at 23 .0 °C.

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H20/ACN solvent occurring in the background of the hydroxyl radical bleaching of the
MB.

After the 60 minute remediation reaction period, the Fenton’s reaction was
quenched with 6 drops (0.27 mL) of 10% NazSO3 solution. Immediately following the
addition of the 10% Nast3, the pH of the reaction mixture increased from
approximately 3.0 to 9.0. No significant change occurred in the temperature of the
reaction mixture. The addition of 10% NazSOg resulted in a change in reaction mixture
appearance to clear and colorless with very fine rust colored (light orange) particles in
suspension. The appearance of rust colored particles can be attributed to the more basic
pH of the reaction mixture during the quenching process, resulting in the conversion of
iron from a hydrated ferrous form to a colloidal ferric form and the formation of ferric
hydroxide (I 4 -16). As shown in Figure 7.1G, the MB dye test of the Fenton’s reaction
mixture quenched with 6 drops of 10% Na2803 solution produced results that closely
resembled the MB result indicated for 50/50 Milli-Q HZO/ACN solvent. The similarity
of the MB dye test results for the quenched reaction mixture and 50/50 Milli-Q
HZO/ACN solvent, as well as the absence of an immediate concentrated white
discoloration (bleaching) at the point of application, indicated that quenching was
complete and no hydroxyl radicals remained.

As a precautionary measure, an additional 2 more drops (0.10 mL) of 10%
Na2803 solution was added to the reaction mixture. Following the addition of the
additional 10% Na2803, the pH and appearance of the reaction mixture remained
unchanged from that observed following the initial addition of 10% NazSO3. As shown

in Figure 7.1H, the MB dye test of the Fenton’s reaction mixture quenched with an

177

additional 2 drops of 10% NaZSO3 solution (a total of 8 drops) produced results that
closely resembled the MB results indicated for 50/50 Milli-Q H20/ACN solvent and the
F enton’s reaction mixture quenched with 6 drops of 10% Na2803 solution, thereby
verifying that quenching was complete.

Since the pH of the reaction mixture was already 9.0, no adjustment of the pH
was required to aid in the removal of iron from the reaction mixture. Since the reaction
mixture was not highly concentrated with precipitate, no centrifugation was performed
prior to filtration. No rinsing of the filtration funnel or reaction vessel with Milli-Q water
was necessary for the filtration of the reaction mixture. The filter was a uniform light
rust color following filtration. The filtrate appeared clear and colorless with no
precipitate. Following adjustment of the filtrate (final Fenton’s remediation solution) to
pH 7, the solution remained clear and colorless with no precipitate.

Throughout the Fenton’s remediation experiment, the amount of water added by
addition of 5.0 mM FeSO4, pH adjustment, addition of 3% H202, quenching, and rinsing
of the filtration funnel was monitored, and an adjusted total H20/ACN ratio by volume
was calculated at the completion of the experiment. The total H20/ACN ratio suggested
the degree of dilution of the sample that occurred during the experiment and was
maintained as constant as possible between the remediation experiments to allow for
comparison of the results. The original HZO/ACN ratio at the start of the remediation
experiment, based on the solution used to dissolve the 4,4'DCBP, was 50/50 or 1.00. At
the completion of the experiment, the calculated adjusted total HZO/ACN ratio for 60

minutes of remediation was 1.12.

178

7.3.2 Dose-Response Bioassay

The final Fenton’s remediation solution was refiigerated at 4 °C until toxicology
bioassays were performed. When the final F enton’s remediation solution was removed
from the refrigerator, the solution appeared clear and colorless with very small white
crystals. The solution was warmed to room temperature (~ 23.0 °C) and sonicated to
achieve complete dissolution of the precipitate prior to use in the bioassay. When test
volumes of the final Fenton’s remediation solution were added to the dishes of confluent
cell cultures, the medium remained clear and colorless with no oily film, precipitate, or
cloudiness. Figure 7.2 presents the dose-response GJIC bioassay results for a volume
range of O to 60 uL of final Fenton’s remediation solution and 30 minutes of incubation.
A gradual decline in GJIC was observed with an increase in test volume. Partial
inhibition of GJIC (F 0C between 0.9 and 0.5) occurred for test volumes greater than 40

uL, and a maximum level of inhibition was attained at 60 uL (F CC = 0.75 :t 0.03).

7.3.3 Time-Response Bioassay

The final Fenton’s remediation solution was refiigerated at 4 °C until toxicology
bioassays were performed. When the final Fenton’s remediation solution was removed
from the refrigerator, the solution appeared clear and colorless with very small white
crystals. The solution was warmed to room temperature (~ 23.0 °C) and sonicated to
achieve complete dissolution of the precipitate prior to use in the bioassay. A 60 uL test
volume was selected for the time-response experiment since it was the highest volume
that caused a substantial amount of inhibition at 30 minutes of chemical exposure. When

test volumes of the final F enton’s remediation solution were added to the dishes of

179

Figure 7.2 Dose-response GJIC bioassay results with a 30 minute incubation time for a
volume range of O to 60 uL of a solution resulting from Fenton’s remediation of 4,4'-
dichlorobiphenyl in 50/50 Milli-Q HzO/ACN solvent. Each data point is representative
of the results for a set of chemically treated triplicates reported as an average FOC i
standard deviation determined at the 95% confidence interval. The Fenton’s reaction
conditions were pH 3.0, temperature 23.0 °C, Fe2+zH202 molar ratio 1:20, initial Fe2+
concentration 0.15 mM, and initial H202 concentration 3 mM. After a 60 minute
remediation reaction period, the F enton’s reaction was quenched with a 10% Na2803
solution (w/v). Following quenching with 10% Na2803, the Fenton’s reaction mixture
pH increased to 9.0 and the temperature remained at 23.0 °C. The reaction mixture was
then filtered through a 1.0 urn glass fiber filter and the filtrate was adjusted to pH 7.

180

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confluent cell cultures, the medium remained clear and colorless with no oily film,
precipitate, or cloudiness. The cell cultures were exposed to the final F enton’s
remediation solution test volume for varying amounts of time ranging from 0 minutes to
1440 minutes (24 hours) followed by GJIC bioassays.

As shown in Figure 7.3, the time-response results for the final Fenton’s
remediation solution indicated partial inhibition of GJIC followed by complete recovery
with an increase in chemical exposure time. Complete recovery of GJIC was assumed to
be attained at a F OC value of 0.97 and greater. A decrease in GJIC was initially
observed with partial inhibition achieved at 20 minutes (FOC = 0.85 i 0.02) and a
maximum level of inhibition at 30 minutes (FOC = 0.78 i 0.02). Between 30 and 240
minutes (a period of 21 0 minutes) recovery of communication without removal of the
chemical was exhibited. Complete recovery of GJIC occurred at 240 minutes (F CC =
1.00 i 0.02) and was maintained through 1440 minutes (FOC = 0.98 i 0.03).

One possible explanation for this partial recovery without removal of the
chemical might be the activation of an autoregulatory pathway. In this case, the chemical
would activate a pathway that would act to inhibit GJIC and then, in an autoregulatory
fashion, another pathway would be activated which would reestablish GJIC. This partial
recovery might also be attributed to the cell’s ability to adapt to the change in conditions
(such as cell homeostasis) due to toxicant exposure that resulted in the initial inhibition.
Finally, partial recovery without removal of the chemical might be a consequence of the
cells metabolizing the chemical into less toxic metabolites.

Since in the time-response experiment, complete recovery of GJIC without

removal of the chemical occurred for the highest test volume that resulted in maximum

182

Figure 7.3 Time-response GJIC bioassay results for a 60 uL test volume of a solution
resulting from Fenton’s remediation of 4,4'-dichlorobiphenyl in 50/50 Milli-Q HzO/ACN
solvent. Time of chemical exposure varied from 0 minutes to 1440 minutes (24 hours).
Each data point is representative of the results for a set of chemically treated triplicates
reported as an average FOC i standard deviation determined at the 95% confidence
interval. Data points after the break correspond to 240 minutes, 480 minutes, 720
minutes, and 1440 minutes of chemical exposure time, respectively. The Fenton’s
reaction conditions were pH 3.0, temperature 23.0 °C, Fe2+zH202 molar ratio 1:20, initial
Fe2+ concentration 0.15 mM, and initial H202 concentration 3 mM. After a 60 minute
remediation reaction period, the Fenton’s reaction was quenched with a 10% Na2803
solution (w/v). Following quenching with 10% Na2803, the Fenton’s reaction mixture
pH increased to 9.0 and the temperature remained at 23.0 °C. The reaction mixture was
then filtered through a 1.0 um glass fiber filter and the filtrate was adjusted to pH 7.

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inhibition of GJIC in the 30 minute does-response bioassay, it was decided that
cytotoxicity and time of recovery bioassays were not required. Complete recovery of
GJIC without removal of the chemical is an indication that the final Fenton’s remediation
solution did not have a cytotoxic effect. Since complete recovery was achieved without
removal of the chemical, time of recovery experiments involving recovery following

removal of the chemical were unnecessary.

7.4 Conclusions

F enton’s remediation of a solution of 4,4'DCBP in 50/50 Milli-Q HZO/ACN (by
volume) was performed. Methylene blue (MB) dye tests were performed during the
Fenton’s reaction (unquenched) to verify the formation of hydroxyl radicals and during
the quenching process to verify completion of quenching (absence of hydroxyl radicals).
The quenched and unquenched MB dye test results for Fenton’s remediation of a solution
of 4,4'DCBP in 50/50 Milli-Q HzO/ACN (Figure 7.1) were identical in appearance to
those observed for Fenton’s remediation of 50/50 Milli-Q HzO/ACN solvent (Figure 6.4).
It can therefore be concluded that the ability of the MB dye test to indicate the presence
of hydroxyl radicals does not appear to be influenced by the presence of 4,4'DCBP.

For the 30 minute dose-response GJIC bioassay of the final Fenton’s remediation
solution, a gradual decline in GJIC was observed with an increase in test volume. Partial
inhibition of GJIC occurred for test volumes greater than 40 uL, and a maximum level of
inhibition was attained at 60 uL (FOC = 0.75 i 0.03). For the time-response GJIC
bioassay of the final Fenton’s remediation solution (60 uL test volume) a maximum level

of inhibition was attained at 30 minutes (FOC = 0.78 i 0.02) of chemical exposure

185

followed by complete recovery of GJIC without removal of the chemical by 240 minutes
of chemical exposure. The complete recovery of GJIC without removal of the chemical
observed for the final Fenton’s remediation solution was similar to the partial recovery of
GJIC without removal of the chemical observed for the potential remediation byproducts
2,2'-biphenyldiol (2,2'BP) and 4,4'-dichloro-3 -biphenylol (4,4'DC3 BP) studied in
Chapter 2.3.4. Since F enton’s remediation of 4,4'DCBP would result in the addition of a
hydroxyl group to nonhalogenated sites and dechlorination reactions are less likely to
occur (2), 4,4'DC3BP is a more favorable remediation byproduct than 2,2'BP. As
discussed in detail in Chapter 2, among the potential remediation byproducts
investigated, 4,4'DC3BP was most inhibitory to GJIC at the lowest dose (F OC = 0.46 i
0.03 at 40 uM) and exhibited cytotoxicity at doses greater than 40 uM. Since the
concentrations of the remaining 4,4'DCBP and remediation byproducts in the final

F enton’s remediation solution were unknown, only the toxicity of the solution as a whole
is considered here. However, the presence of 4,4'DC3BP as a remediation byproduct
might have contributed to the inhibitory effects and complete recovery observed for the
final Fenton’s remediation solution. The inhibitory effects of 4,4’DC3BP previously
observed in Chapter 2 might have been attenuated by its presence at a lower
concentration as a component of the final Fenton’s remediation solution. Whereas only
partial recovery of GJIC without removal of the chemical was achieved for 4,4'DC3BP in
the potential remediation byproduct studies of Chapter 2, complete recovery of GJIC
without removal of the chemical could have been attained at lower concentrations of
4,4'DC3BP in the final Fenton’s remediation solution. The final Fenton’s remediation

solution was determined to be more toxic (inhibitory to GJIC) than the parent PCB,

186

4,4'DCBP, which exhibited very slight to no inhibition of GJIC for the dose-response
experiments for incubation times of 30 minutes, 2 hours, 6 hours, and 24 hours in Chapter

3.

187

7.5 References

1.

10.

11.

12.

Hemer, H.A.; Trosko, J .E.; Masten, S.J. The Epigenetic Toxicity of Pyrene and
Related Ozonation Byproducts Containing an Aldehyde Functional Group. Environ.
Sci. Technol. 2001, 35 (17), 3576-3583.

Sedlak, D.L.; Andren, A.W. Aqueous-Phase Oxidation of Polychlorinated Biphenyls
by Hydroxyl Radicals. Environ. Sci. Technol. 1991, 25 (8), 1419-1427.

Sato, C.; Leung, S.W.; Bell, H.; Burkett, W.A.; Watts, R.J. Decomposition of
Perchloroethylene and Polychlorinated Biphenyls with F enton 's Reagent (Chapter

I 6). Emerging Technologies in Hazardous Waste Management III (ACS Symposium
Series), American Chemical Society: Atlanta, GA, 1991.

Micaroni, R.C.C.M.; Bueno, M.I.M.S.; Jardim, W.F. Degradation of Acetonitrile
Residues Using Oxidation Processes. J. Braz. Chem. Soc. 2004, 15 (4), 509-513.

Satoh, A.Y.; Trosko, J .E.; Masten, SJ. Epigenetic Toxicity of Hydroxylated
Biphenyls and Hydroxylated Polychlorinated Biphenyls on Normal Rat Liver
Epithelial Cells. Environ. Sci. Technol. 2003, 3 7 (12), 2727-2733.

Trapido, M; Goi, A. Degradation of nitrophenols with the Fenton reagent. Proc. Est.
Acad. Sci. Chem. 1999, 48(4), 163-173.

Satoh, A.Y.; Trosko, J .E.; Masten, S.J. Methylene Blue Dye Test for Rapid
Qualitative Detection of Hydroxyl Radicals Formed in a Fenton’s Reaction Aqueous
Solution. Environ. Sci. T echnol. 2007, 41 (8), 2881-2887.

Mohamadin, A.M. Possible role of hydroxyl in the oxidation of dichloroacetonitrile
by F enton-like reaction. J. Inorg. Biochem. 2001, 84 (1-2), 97-105.

Trapido, M. Tallinn Technical University, Tallinn, Estonia. Personal Communication,
2002.

Trosko, J .E. Michigan State University, East Lansing, MI. Personal Communication,
August 2003.

Luster-Teasley, S.L.; Yao, J.J.; Hemer, H.A.; Trosko, J .E.; Masten, SJ. Ozonation of
Chrysene: Evaluation of Byproduct Mixtures and Identification of Toxic Constituent.
Environ Sci. Technol. 2002, 36(5), 869-876.

El-Fouly, M.H.; Trosko, J .E.; Chang, C.C. Scrape-Loading and Dye Transfer: A rapid

and simple technique to study gap junctional intercellular communication. Exp. Cell
Res. 1987, 168, 422-430.

188

13. Wilson, M.R.; Close, T.W.; Trosko, J .E. Cell Population Dynamics (Apoptosis,
Mitosis, and Cell-Cell Communication) during Disruption of Homeostasis. Exp. Cell
Res. 2000, 254, 257-268.

14. Lindsey, M. E.; Tarr, M. A. Quantitation of hydroxyl radical during F enton oxidation
following a single addition of iron and peroxide. Chemosphere 2000, 41 (3), 409-417.

15. Arnold, S. M.; Hickey, W. J .; Harris, R. F. Degradation of atrazine by Fenton’s
reagent: condition optimization and product quantification. Environ. Sci. Technol.
1995, 29(8), 2083-2089.

16. Pratap, K.; Lemley, A. T. Fenton electrochemical treatment of aqueous atrazine and
metolachlor. J Agric. Food Chem. 1998, 46(8), 3285-3291.

189

Chapter 8

Time Course Studies: Fenton’s Remediation of 4,4'-Dichlorobiphenyl in
50/50 Milli-Q Water/Acetonitrile and Disappearance of Parent PCB

8.1 Introduction

One method for evaluating the effectiveness of a remediation process is to
measure the disappearance of the parent compound. Current remediation practices often
emphasize the disappearance of the parent compound to at or below regulatory limits, but
often disregard the importance of reducing the overall toxicity. Since remediation
byproducts can exhibit equal or greater toxicity than the parent compound, it is important
to consider in a remediation process not only the removal of the parent compound, but
also the toxicological impact of the remediation byproducts (I). This chapter discusses
the disappearance of 4,4'-dichlorobiphenyl as a result of Fenton’s remediation.
Toxicological analyses of the solution resulting fiom Fenton’s remediation of 4,4'-
dichlorobiphenyl are presented in Chapter 7.

In environmental applications, PCB concentrations have often been estimated by
comparing retention patterns and gas chromatography/ electron capture detection
(GC/ECD) response factors from approximated mixtures of Aroclor standards or PCB
congeners (2, 3). The electron capture detector (ECD) is highly sensitive to molecules
containing electronegative functional groups such as halogens, peroxides, quinones, and
nitro groups, but insensitive to functional groups such as amines, alcohols, and
hydrocarbons (4). The selective nature of the ECD response towards halogen containing
compounds has made it one of the most widely used detectors for environmental samples

containing chlorinated contaminants, such as chlorinated insecticides and polychlorinated

190

biphenyls (4). The more halogenated the molecule, the more sensitive the ECD is to
detecting that compound. Therefore, the ECD is more sensitive to detecting highly
chlorinated compounds. However, the ECD is capable of detecting compounds with
fewer chlorines, if a sufficiently high concentration of the compound is present.

In this section of research, Fenton’s remediation of solutions of 4,4'-
dichlorobiphenyl (4,4'DCBP) in 50/50 Milli-Q HZO/ACN was performed for 0, 15, 30,
and 60 minutes. Acetonitrile (ACN) was added as part of the solvent to increase the
solubility of the 4,4'DCBP. It has been shown that the presence of the acetonitrile does
not inhibit the Fenton’s reaction, as it is not appreciably reactive with hydroxyl radicals
(5), and in toxicology studies it does not affect GJIC at low concentrations (6). To
prepare the remediation mixtures for GC/ECD analysis, an isooctane liquid-liquid
extraction method was developed that resulted in greater than ninety percent efficiency of
recovery of 4,4'DCBP. The extracted samples were analyzed by GC/ECD to quantitate
the disappearance of the parent PCB, 4,4'DCBP, over the period of remediation. In
addition, retention times were determined for the chlorinated potential remediation
byproducts previously investigated in toxicology studies (Chapter 2) and compared to the

retention patterns of the extracted samples.

8.2 Experimental Section
8.2.1 Chemicals

The parent PCB, 4,4'-dichlorobiphenyl (99% purity), and chlorinated potential
remediation byproducts, 3-chloro-2~biphenylol (95% purity) and 4,4 '-dichloro—3-

biphenylol (95% purity), were all purchased fi'om Chem Service Inc. (West Chester, PA).

191

Acetonitrile (99.8% purity) was purchased from EM Science (Gibbstown, NJ). Isooctane
(99.7% purity) was purchased fiom Burdick & Jackson (Muskegon, MI). Methylene blue
dye (3,7-bis(dimethylamino)-phenothiazin—5-ium chloride) (97% purity) was purchased
from Fluka (Buchs, Switzerland). Thirty-percent hydrogen peroxide (HzOz)
(unstabilized), iron(II) sulfate heptahydrate (F eSO4-7H20) (99% purity), and sodium
sulfite (Na2SO3) (anhydrous, 98% purity) were purchased from Sigma-Aldrich (St. Louis,
MO). Sulfuric acid (HzSO4) (96% purity) and sodium hydroxide (NaOH) pellets (99%
purity) were purchased from J .T. Baker (Phillipsburg, NJ). Throughout this chapter and
dissertation, Milli—Q water was obtained fiom a Milli-Q Ultrapure Water Purification

System (System Type ZMQS6VFOY) purchased fiom Millipore Corp. (Bedford, MA).

8.2.2 Methods
8.2.2.1 Gas Chromatography/Electmn Capture Detector (GC/ECD)

GC/ECD was performed using a Perkin-Elmer AutoSystem Gas Chromatograph
(GC) equipped with an electron capture detector (ECD) (Norwalk, CT). A DB5 (30 m
length x 0.25 mm id. x 0.25 pm film thickness) fused silica capillary column (J&W
Scientific, Folsom, CA) with phase composition cross linked/surface bonded 5% phenyl,
95% methylpolysiloxane was used for the GC separation. The autosampler was set to
inject a sample volume of 1.0 uL. A splitless injector was used with a column head
pressure of 15 psi using nitrogen as the carrier gas, producing a flow rate of 1 mL/min.
The initial column temperature was held for 1 minute at 50 °C and then ramped at 10
°C/minute to 250 °C for a total run time of 21.00 minutes. Isooctane was used as a blank

and was run in the GC/ECD prior to each set of samples.

192

8.2.2.2 Calibration Curve

For each set of GC experiments, a calibration curve was determined for
4,4 'DCBP/isooctane to develop a mathematical relationship between the concentration
(ppm) and the area under the curve (uV*sec). Calibration standards, 20, 40, 80, and 160
ppm 4,4'DCBP/isooctane, were prepared and GC/ECD was performed. Using the
GC/ECD results, a graph of area vs. prepared concentration was prepared and the
calibration curve equation was determined by linear regression. Using the areas provided
by the GC/ECD for liquid-liquid isooctane extraction samples of the Fenton’s
remediation mixtures, this equation was used to determine the concentrations of

4,4 'DCBP/isooctane.

8.2.2.3 Percent Efficiency of Recovery by Isooctane Extraction

The percent efficiency of recovery of 4,4'DCBP by isooctane extraction was
studied to (1) ensure the isooctane liquid-liquid extraction method resulted in greater than
ninety percent efficiency of recovery of 4,4'DCBP and (2) determine the maximum
allowable percent of ACN in the dilution water during the extraction procedure without
adversely affecting the efficiency. The extraction procedure involved a combination of
Milli-Q water and isooctane. Milli-Q water was added to dilute the acetonitrile in the
Fenton’s remediation mixture. Consistent with EPA Method 8082 (3), isooctane was
selected as the extraction solvent. In the extraction mixture, the ACN and Milli-Q water
formed the bottom layer, while the 4,4'DCBP and isooctane formed the top layer
(extracted sample). Although numerous combinations of Milli-Q water and isooctane

were evaluated for potential extraction procedures, the efficiency of the extraction

193

 

 

procedure finally selected for studying the disappearance of the parent PCB is primarily
discussed here.

A 415 ppm (1.86 mM) stock solution of 4,4'DCBP/ACN was prepared as a
sample for extraction. Although the extraction procedure was conducted in a 60 mL
borosilicate extraction vial, the total volume of the extraction mixture was 63 mL. By
utilizing the narrow neck of the vial for the excess volume beyond 60 mL, the isooctane
extraction layer could be clearly identified and easily removed. In separate vials, 800,
400, 200, and 100 uL of the 4,4'DCBP/ACN stock solution was added corresponding to
the calculated expected extraction concentrations of 166, 83, 41.5, and 20.75 ppm
4,4'DCBP/isooctane, which assumed 100% efficiency of recovery by isooctane
extraction. The extraction process was performed in two parts, each part involving the
addition of Milli-Q water to dilute the ACN fiom the sample, the addition of isooctane to
extract the 4,4’DCBP from the sample, and shaking/vortexing of the sample. The first
part of the extraction process involved the addition of 31 mL of Milli-Q water followed
by shaking/vortexing, and then the addition of 1 mL of isooctane followed by
shaking/vortexing. The second part of the extraction process involved the addition of 30
mL of Milli-Q water followed by shaking/vortexing, and then the addition of 1 mL of
isooctane followed by shaking/vortexing. The extraction mixture was then allowed to
rest until a distinct clear top and bottom layer were visible. When removing the extracted
sample for the GC/ECD, it was important to ensure that it contained no ACN or water.
To improve visualization of the top layer of the extraction mixture, consisting of the
extracted 4,4'DCBP and isooctane, this layer was carefully transferred by micropipette to

a 4 mL borosilicate culture tube. Approximately 1 mL was transferred from the surface

194

of the solution in the culture tube to a GC sample vial. GC/ECD was performed for each
extracted sample using the procedure described previously.

From the areas determined by the GC/ECD, the true extraction concentrations of
4,4’DCBP/isooctane were calculated from the calibration curve. To calculate the percent
efficiency of recovery by isooctane extraction, the true extraction concentration of
4,4’DCBP/isooctane was divided by the expected extraction concentration of
4,4’DCBP/isooctane and then multiplied by 100. An efficiency of recovery greater than
ninety percent was preferred, since this would mean that greater than ninety percent of
the 4,4 'DCBP was extracted by the isooctane and no mathematical adjustment of the true
extraction concentration would be required. Assuming the volume of 4,4'DCBP/ACN
stock solution was primarily ACN and was not significant compared to the volume of
dilution water, the percent of ACN in the dilution water was calculated as the volume of
stock solution added to the vial divided by the total volume of Milli-Q water added to

dilute the ACN (61 mL) and then multiplied by 100.

8.2.2.4 Fenton’s Remediation with Varying Reaction Times

Fenton’s remediation of solutions of 4,4'-dichlorobiphenyl (4,4'DCBP) in 50/50
Milli-Q HZO/ACN (by volume) was performed for 0, 15, 30, and 60 minutes. The
initiation of remediation was defined as the moment 3% H202 was added. Since it is
impossible to obtain a true zero time of remediation, as this would require
instantaneously quenching the reaction following its initiation, “0 minutes” of
remediation was interpreted as the remediation procedure just prior to the addition of 3%

H202. For each of the non-zero remediation times, an Fe2+zH202 molar ratio of 1:20 was

195

used for the Fenton’s reaction. The initial concentrations of Fe2+ and H202 in the reaction
mixture were 0.15 mM and 3 mM, respectively, which are within the ranges previously
used by Trapido et al. (7). The volume of 5.0 mM FeSO4 and 3% H202 added to the
reaction mixture was calculated to reflect the assumption that this addition caused a
significant change in the overall reaction mixture volume. The amount of water added by
addition of 5.0 mM F eSO4, pH adjustment, addition of 3% H202, quenching, and rinsing
of the filtration fimnel was monitored throughout the experiment, and an adjusted total
HzO/ACN ratio was calculated at the completion of the experiment. In this calculation,
the volumes of 5.0 mM FeSO4, 3% H202, 10% NaZSO3, 0.5 M H2804, and 1M NaOH
added were assumed to be 100% water. The total H20/ACN ratio suggested the degree
of dilution of the sample that occurred during the experiment. In each experiment, this
ratio was maintained as constant as possible to allow for comparison of the results. The
pH and temperature were monitored throughout the experiments.

Methylene blue (MB) dye tests were performed during the Fenton’s reaction to
verify the formation of hydroxyl radicals, during the quenching process to verify
completion of quenching (absence of hydroxyl radicals), and during the “0 minutes” of
remediation experiment to verify the absence of hydroxyl radicals. Forty microliters of
liquid sample was placed dropwise onto the center of the MB dyed section of a test strip,
allowing for absorption between drops. The MB dye tests were compared against the test
strip tested with 50/50 Milli-Q H20/ACN solvent, which is characterized by a diffuse
discoloration of the MB, spreading out/emanating from the point of application, with a
dark blue band surrounding the area of discoloration. MB dye tests indicating the

absence of hydroxyl radicals in a reaction mixture with a 50/50 Milli-Q HzO/ACN

196

solvent were identified by a resemblance to the MB dye test results for 50/50 Milli-Q
HzO/ACN solvent alone. Discoloration of the MB dye, due to the presence of hydroxyl
radicals in a reaction mixture with a 50/50 Milli-Q H20/ACN solvent, was indicated by
an immediate concentrated white discoloration (bleaching) at the point of application
surrounded by diffuse discoloration of the MB to light blue, spreading out/emanating
from the point of application, with a dark blue band surrounding the discolored area.

Each experiment involved the remediation of approximately 8.0 mg 4,4'DCBP. A
50 mL centrifuge tube with a vaned tube stir bar was used as the reaction vessel. The
4,4'DCBP was dissolved in 50/50 Milli-Q HZO/ACN (by volume), in a step-wise
procedure, to obtain a final concentration of 2 mM 4,4'DCBP (the final concentration of
the test solution used in the 4,4’DCBP toxicology study). To improve the dissolution of
4,4'DCBP, it was first dissolved in the ACN portion of the 50/50 Milli-Q HZO/ACN
solvent, and then the Milli-Q H20 portion was added in 1 mL increments. The reaction
mixture was sonicated following each addition of the solvent. A 5.0 mM F eSO4 stock
solution was prepared from FeSO4—7HzO and Milli-Q water. The 5.0 mM FeSO4 stock
solution was then added to the reaction mixture at the calculated volume to achieve a
final concentration of 0.15 mM Fe”. For optimal Fenton’s reaction efficiency, the
reaction mixture was adjusted to pH 3 with 0.5 M H2804 and/or 1 M NaOH. The pH was
adjusted/monitored using a 720A plus pH/ISE meter with an 8102 BNU Ross Ultra
Combination pH electrode (ThermoOrion, Beverly, MA).

For “0 minutes” of remediation, there was no addition of 3% H202; however,
methylene blue (MB) dye tests were performed on 40 uL samples of the reaction mixture,

Milli-Q water, and 50/50 Milli-Q H20/ACN solvent. The reaction mixture was then

197

filtered through a 1.0 pm glass fiber filter, using a vacuum filtration unit, into a large
culture tube inserted into a 1000 mL Erlenmeyer side-arm filtration flask. Milli-Q water
was used for rinsing during filtration only if the reaction mixture touched the sides of the
funnel or there was residue in the culture tube. To maintain the conditions and chemical
species similar to what would exist prior to remediation initiation, the filtered reaction
mixture was not readjusted to pH 7. The filtered reaction mixture was transferred to an
amber, 60 mL Boston round bottle with a TFE closure and kept at room temperature for
the isooctane extraction procedure.

For each of the non-zero remediation times, following the adjustment of the
reaction mixture to pH 3, the F enton’s reaction was initiated by the addition of 3% H202,
prepared from 30% unstabilized H202 and Milli-Q water, to the reaction mixture to
obtain an initial concentration of 3 mM H202. Since stabilizing agents (hydroxyl radical
scavengers) in commercial H202 might affect the results (8), only H202 devoid of
stabilizing agents was used. In order to prevent localized reactions (that might occur
when a small volume of very concentrated solution is added to a reaction mixture), 3%
H202 rather than 30% H202 was used to initiate the reaction. The remediation reaction
was allowed to occur for 15, 30, or 60 minutes with occasional sonication of the reaction
mixture. To test, qualitatively, the production of hydroxyl radicals during the reaction,
MB dye tests were performed on the unquenched reaction mixture at varying times
during the remediation. In addition, MB dye tests were performed on 40 uL samples of
Milli-Q water and 50/50 Milli-Q HZO/ACN solvent.

After the specified remediation reaction period, the F enton’s reaction was

quenched with a 10% Na2803 solution (w/v), prepared fi'om Na2803 and Milli-Q water.

198

Trapido et al. (7, 9) recommended quenching by the addition of 2 to 3 drops of 10%
Na2SO3 solution for every 10 mL of reaction mixture. A 0.5 mL glass pipette was used
to administer the drops to the reaction mixture. To verify, qualitatively, that quenching
was complete, 5 minutes after the addition of the 10% Na2803 solution, a MB dye test
was performed. An additional aliquot of 10% Na2SO3 solution was added to the reaction
mixture, and the MB dye test was repeated until an absence of hydroxyl radicals in the
reaction mixture was indicated (quenching was complete). To aid in the removal of iron
from the reaction mixture by precipitation of F e3”, following quenching, the reaction
mixture was adjusted to pH 9 with 0.5 M H2304 and/or 1 M NaOH. If a large amount of
precipitation in the reaction mixture was observed, the solution was centrifuged at 1200
rpm for 10 minutes and the supernatant was retained for filtration. The reaction mixture
or supernatant was then filtered through a 1.0 pm glass fiber filter, using a vacuum
filtration unit, into a large culture tube inserted into a 1000 mL Erlenmeyer side-arm
filtration flask. Milli-Q water was used for rinsing during filtration only if the reaction
mixture touched the sides of the fimnel or there was residue in the centrifuge tube. The
filtered reaction mixture was transferred to a clean 50 mL centrifuge tube containing a
vaned tube stir bar. To parallel the F enton’s remediation toxicology studies (in which the
reaction mixture pH was neutralized for the cells in the GJIC bioassays), and maintain
similar conditions and chemical species, the filtered solution was then adjusted to pH 7
with 0.5 M H2804 and/or 1 M NaOH. This final solution was transferred to an amber, 60
mL Boston round bottle with a TFE closure and kept at room temperature for the

isooctane extraction procedure. The assumption was made that the filtration process

199

performed during the F enton’s remediation experiment did not remove remaining

4,4'DCBP and byproducts generated during the reaction.

8.2.2.5 Isooctane Extraction Procedure

For GC/ECD analysis, two solutions of different concentrations were prepared by
isooctane extraction fi'om each of the final Fenton’s remediation solutions stored in the
Boston round bottles. Since it is unknown prior to GC/ECD analysis what the true
concentration of 4,4'DCBP is in each of the final Fenton’s remediation solutions, for the
purpose of calculations the assumption was made that no remediation had occurred and
the concentration of 4,4'DCBP was the same as at the start of the Fenton’s reaction (2
mM or 446 ppm). This assumption also prevented the “overloading” of the GC column,
which can result from the injection of a sample that is too concentrated. In addition, for
the purpose of calculations, the assumption was made that there would be 100%
efficiency of recovery by isooctane extraction. Based on these assumptions, the two
calculated “concentrations” of 4,4 'DCBP in the prepared isooctane extraction solutions
were 156.1 and 78.05 ppm (the quotes indicating that these are not the true
concentrations, but rather calculated concentrations that would be expected based on the
assumptions mentioned above). For each extraction pair, 700 uL and 350 uL of the final
F enton’s remediation solution were placed in separate 60 mL borosilicate extraction
vials, corresponding to the isooctane extraction solution “concentrations” 156.1 ppm and
7 8.05 ppm, respectively.

The extraction process was performed in two parts, each part involving the

addition of Milli-Q water to dilute the ACN from the sample, the addition of isooctane to

200

extract the 4,4'DCBP from the sample, and shaking/vortexing of the sample. The first
part of the extraction process involved the addition of 31 mL of Milli-Q water followed
by shaking/vortexing, and then the addition of 1 mL of isooctane followed by
shaking/vortexing. The second part of the extraction process involved the addition of 30
mL of Milli-Q water followed by shaking/vortexing, and then the addition of 1 mL of
isooctane followed by shaking/vortexing. The extraction mixture was then allowed to
rest until a distinct clear top and bottom layer were visible. In the extraction mixture, the
ACN and Milli-Q water formed the bottom layer, while the 4,4'DCBP and isooctane
formed the top layer (extracted sample). When removing the extracted sample for the
GC/ECD, it was important to ensure that it contained no ACN or water. To improve
visualization of the top layer of the extraction mixture, consisting of the extracted
4,4'DCBP and isooctane, this layer was carefully transferred by micropipette to a 4 mL
borosilicate culture tube. Approximately 1 mL was transferred from the surface of the
solution in the culture tube to a GC sample vial. GC/ECD analysis was performed in

triplicate for each extracted sample using the procedure described previously.

8.2.2.6 Fenton’s Remediation Solution Concentration by Back-Calculation

The concentrations of the final Fenton’s remediation solutions were determined
through a process of back-calculation fi'om the GC/ECD results. From the areas
determined by the GC/ECD, the true extraction solution concentrations of
4,4'DCBP/isooctane (ppm) were calculated from the calibration curve. Since ppm can be

defined as pg divided by mL, assuming 100% isooctane extraction efficiency, the mass

(pg) of 4,4'DCBP extracted from the sample of final Fenton’s remediation solution

201

placed in the extraction vial was determined by multiplying the true extraction solution
concentration of 4,4'DCBP/isooctane (ppm) by the 2 mL of isooctane used for extraction.
The concentration of the final F enton’s remediation solution was then determined by
dividing the extracted mass (pg) of 4,4'DCBP by the volume of final Fenton’s
remediation solution placed in the extraction vial (mL).

Since these calculations assumed 100% isooctane extraction efficiency, the actual
percent isooctane extraction efficiency was determined from the percent of ACN in the
dilution water and the results of the “Percent Efficiency of Recovery by Isooctane
Extraction” experiment. The solvent used in the Fenton’s remediation of 4,4'DCBP was
50/50 Milli-Q H20/ACN by volume. Assuming that the volume of ACN was not
changed significantly by the remediation process, and the total volume of solvent in the
sample of final Fenton’s remediation solution was not significant compared to the volume
of dilution water in the extraction process, the percent of ACN in the dilution water was
calculated as the volume (mL) of ACN added to the extraction vial (50% of the volume
of final F enton’s remediation solution placed in the extraction vial) divided by the total
volume of Milli-Q water added to dilute the ACN during the extraction process (61 mL)
and multiplied by 100. From the “Percent Efficiency of Recovery by Isooctane
Extraction” experiment, a mathematical relationship was developed between the percent
of ACN in the dilution water and the isooctane extraction efficiency. Using this
mathematical relationship, the isooctane extraction efficiency was determined from the
percent of ACN in the dilution water in the isooctane extraction of the final Fenton’s
remediation solutions. If the actual percent isooctane extraction efficiency was greater

than ninety percent, no mathematical adjustment of the true extraction solution

202

concentration (and hence the final Fenton’s remediation solution concentration) of
4,4'DCBP was required.

Since two solutions of different concentrations were prepared by isooctane
extraction from each of the final F enton’s remediation solutions and GC/ECD analysis
was performed in triplicate for each extracted sample, a total of six calculated
concentrations were determined for each of the final Fenton’s remediation solutions.
From these six concentrations, the average concentration and standard deviation for each
of the final F enton’s remediation solutions were determined. Based on the concentration
of 4,4'DCBP at the start of the Fenton’s reaction (446 ppm) and the average
concentration of the final Fenton’s remediation solutions, the average percent of

4,4'DCBP remaining in the final Fenton’s remediation solutions were determined.

8.2.2.7 Retention Times for Chlorinated Potential Remediation Byproducts

In chapter 2, the toxicity of six characteristic potential remediation byproducts
resulting fi'om the Fenton’s remediation of PCBs were examined. The potential
remediation byproducts, hydroxylated biphenyls and hydroxylated polychlorinated
biphenyls, included 2-biphenylol, 3-biphenylol, 2,2'-biphenyldiol, 3,3 '-biphenyldiol, 3-
chloro-2-biphenylol, and 4,4'—dichloro-3 -biphenylol. In order to attempt to identify the
presence of these potential remediation byproducts in the final Fenton’s remediation
solutions, GC/ECD was performed on stock solutions to determine the retention times for
the potential remediation byproducts. Since the ECD response is selective towards
halogen containing compounds, only chlorinated remediation byproducts could be

detected. Therefore, the retention times were determined for only 3-chloro-2-biphenylol

203

and 4,4'-dichloro-3 -biphenylol. Two stock solutions, 220 ppm 3-chloro-2-
biphenylol/isooctane and 200 ppm 4,4'-dichloro-3-biphenylol/isooctane, were prepared
and approximately 1000 pL of each was transferred to GC sample vials. GC/ECD
analysis was performed for each stock solution sample using the procedure described
previously. The retention times of 3-chloro-2-biphenylol and 4,4'-dichloro-3-biphenylol

were determined from the resultant gas chromatograms.

8.3 Results and Discussion
8.3.1 Retention Times for Chlorinated Potential Remediation Byproducts

Although toxicology studies were performed for six potential remediation
byproducts, the retention times were determined for only 3-chloro-2-biphenylol and 4,4'-
dichloro-3-biphenylol, since only the chlorinated compounds could be detected by the
ECD. As shown in Figure 8.1, the gas chromatogram for the blank (isooctane) revealed
no significant peaks, except for small peaks of impurities. The gas chromatograms for
the 220 ppm 3-chloro-2-biphenylol/isooctane and 200 ppm 4,4'-dichloro-3-
biphenylol/isooctane stock solutions are shown in Figures 8.2 and 8.3, respectively. The
retention times for 3-chloro-2-biphenylol and 4,4'-dichloro-3-biphenylol were 14.21

minutes and 17.03 minutes, respectively.

8.3.2 Percent Efficiency of Recovery by Isooctane Extraction
As described previously, four different volumes of 4,4'DCBP/ACN stock solution
were extracted through a process involving Milli-Q water and isooctane. Immediately

following the extraction process, the extraction mixture was a white emulsion. Following

204

Figure 8.1 Gas chromatogram for the blank (isooctane).

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a rest period, a distinct clear top and bottom layer partially separated by a thin, white ring
of froth, along the perimeter of the extraction vial, were visible in the extraction mixture.
The top layer dipped through the center of the froth ring, but remained distinct from the
bottom layer. In the extraction mixture, the ACN and Milli-Q water formed the bottom
layer, while the 4,4'DCBP and isooctane formed the top layer (extracted sample).

The gas chromatogram for the blank (isooctane) revealed no significant peaks,
except for small peaks of impurities. From the gas chromatograms for the calibration
curve, the retention time (average i standard deviation) for 4,4'DCBP was determined to
be 15.30 i- 0.05 minutes. Similarly, the gas chromatograms for the extracted samples
indicated a retention time (average 3: standard deviation) of 15.30 i 0.09 minutes for the
4,4’DCBP peak. For each of the gas chromatograms a small peak was also evident at a
retention time close to 12 minutes; however, since this peak also was present in the gas
chromato gram for the blank (isooctane), it can be attributed to impurities in the isooctane.

Figure 8.4 is a comparison of the isooctane extraction GC/ECD results, based on
the expected extraction concentrations, and the calibration curve. The calibration curve
equation determined by linear regression was y = 15 964x —77522 with an R2 = 0.9949.
The linear regression equation of the relationship of the isooctane extraction GC/ECD
results and the expected extraction concentrations was y = 10455x + 138470 with an R2 =
0.9807. For the isooctane extraction, the areas corresponding to the expected extraction
concentrations 20.75, 41.5, and 83 ppm 4,4'-DCBP/isooctane closely corresponded with
the calibration curve. However, the area corresponding to the expected extraction
concentration of 166 ppm 4,4'DCBP/isooctane differed significantly from the calibration

curve, indicating a lower percent efficiency of recovery. From the GC/ECD isooctane

211

Figure 8.4 Comparison of the isooctane extraction GC/ECD results for efficiency of
recovery of 4,4'-dichlorobiphenyl and the calibration curve for 4,4'DCBP/isooctane. The
isooctane extraction GC/ECD results were graphed based on the expected extraction
concentrations.

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extraction areas, the true extraction concentrations of 4,4 ’DCBP/isooctane were
calculated from the calibration curve linear regression equation. The true extraction
concentrations were then compared to the expected extraction concentrations to
determine the percent efficiency of recovery by isooctane extraction.

Figure 8.5 compares the relationships between the percent efficiency of recovery
of 4,4'DCBP by isooctane extraction, the calculated expected extraction concentrations
based on the original volumes of 4,4’DCBP/ACN stock solution prepared for extraction,
and the percent of ACN in the dilution water of the extraction process. Since higher
calculated expected extraction concentrations resulted from higher volumes of
4,4'DCBP/ACN stock solution prepared for extraction, the percent of ACN in the dilution
water of the extraction process increased with an increase in the expected extraction
concentration. As the percent of ACN in the dilution water of the extraction process
increased, there was a coinciding decrease in the percent efficiency of recovery of
4,4'DCBP by isooctane extraction. Greater than ninety percent efficiency of recovery of
4,4 'DCBP by isooctane extraction was achieved when the percent of ACN in the dilution
water during the extraction procedure was less than 0.66%. At a level of 1.31% ACN in
the dilution water, corresponding to an expected extraction concentration of 166 ppm
4,4'DCBP/isooctane, the efficiency of recovery was only 71.50%.

The detrimental effect of a high percent of ACN in the dilution water on the
percent efficiency of recovery was further emphasized by previous experiments (results
not shown) in which only 39 mL of Milli-Q water was added during the extraction
process rather than 61 mL. In contrast to the results fiom the addition of 61 mL of Milli-

Q water during the extraction process to dilute the ACN, when 39 mL of Milli-Q water

214

Figure 8.5 Comparison of the relationships between the percent efficiency of recovery of
4,4'DCBP by isooctane extraction, the calculated expected extraction concentrations
based on the original volumes of 4,4 ’DCBP/ACN stock solution prepared for extraction,
and the percent of ACN in the dilution water of the extraction process.

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was added instead, therefore resulting in a higher percent of ACN in the dilution water,
the efficiency of recovery was less than ninety percent for the same expected extraction
concentrations at which greater than ninety percent efficiency had been observed. Hence,
the addition of Milli-Q water as a means of diluting the ACN in the extraction process,
thereby decreasing the percent of ACN in the dilution water, is essential to obtaining an

acceptable efficiency of recovery of 4,4'DCBP by isooctane extraction.

8.3.3 Fenton’s Remediation of 4,4'-Dichlorobiphenyl and Extraction

F enton’s remediation of solutions of 4,4’DCBP in 50/50 Milli-Q H20/ACN (by
volume) was performed for “0”, 15, 30, and 60 minutes. To improve the dissolution of
4,4'DCBP, for each remediation, the 4,4'DCBP was dissolved in 50/50 Milli-Q
H20/ACN (by volume) in a step-wise procedure to obtain the final concentration. When
the 4,4'DCBP was first dissolved in the ACN portion of the 50/50 Milli-Q H20/ACN
solvent, the resulting solution was clear and colorless with no precipitate. The solution
appearance remained unchanged during the addition of the Milli-Q H20 portion of the
50/50 Milli-Q H20/ACN solvent until approximately 5.0 mL of Milli-Q H20 had been
added. Further addition of Milli-Q H20 resulted in a light milky white solution. As the
remaining Milli-Q H20 portion was added in 1000 pL increments, increasing amounts of
sonication were necessary in order to maintain complete dissolution. Following the
addition of 5.0 mM F e804 and pH adjustment, the reaction mixture was clear and
colorless with very fine white particles in suspension.

For “0 minutes” of remediation, there was no addition of 3% H202; however, MB

dye tests were performed on 40 pL samples of the reaction mixture, Milli-Q water, and

217

50/50 Milli-Q H20/ACN solvent. As shown in Figure 8.6A, the MB dye test control strip
(no sample added) was homogeneously dark blue in color. When the MB dye test was
performed with Milli-Q water (Figure 8.63), no bleaching or discoloration was observed.
As shown in Figure 8.6C, a MB dye test of 50/50 Milli-Q H20/ACN solvent produced
diffuse discoloration of the MB to light blue, spreading out/emanating from the point of
application, with a dark blue band surrounding the discoloration. As shown in Figure
8.6D, when the MB dye test was performed with the reaction mixture fiom “0 minutes”
of remediation, the result closely resembled the MB result indicated for 50/50 Milli-Q
H20/ACN solvent with only slightly more white diffuse discoloration. The similarity of
the reaction mixture and 50/50 Milli-Q H20/ACN solvent MB dye test results verified the
absence of hydroxyl radicals in the “0 minutes” reaction mixture.

To maintain the conditions and chemical species similar to what would exist prior
to remediation initiation, the reaction mixture was neither adjusted to pH 9 prior to
filtration nor adjusted to pH 7 following filtration. Prior to filtration of the reaction
mixture from “0 minutes” of remediation, sonication was performed; however, the
reaction mixture remained clear and colorless with very fine white particles in
suspension. Following filtration, the reaction mixture appeared clear and colorless with
no precipitate.

For each of the non-zero remediation times, following the adjustment of the
reaction mixture to pH 3, the F enton’s reaction was initiated by the addition of 3% H202.
The remediation reaction was allowed to occur for 15, 30, or 60 minutes with occasional
sonication of the reaction mixture. During the F enton’s reaction remediation period, no

significant change occurred in the solution pH and temperature. The solution remained

218

 

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H20/ACN. Methylene blue dye test results for (A) control (no sample added), (B) 40 pL

of Milli-Q water, (C) 40 pL of 50/50 Milli-Q H20/ACN solvent, and (D) 40 pL of the
reaction rrrixture from “0 minutes” of remediation.

219

clear and colorless with the amount of very fine white particles in suspension decreasing
as the reaction progressed, until no precipitate was apparent, after about 5 minutes.

In addition to performing MB dye tests on 40 uL samples of Milli-Q water and
50/50 Milli-Q H20/ACN solvent, MB dye tests were also performed on 40 uL samples of
the unquenched reaction mixture to verify the production of hydroxyl radicals during the
reaction. Figure 8.7 presents the MB dye test results for 60 minutes of remediation;
however, the results are representative of those observed for the shorter periods of
remediation studied. As shown in Figure 8.7A, the MB dye test control strip (no sample
added) was homogeneously dark blue in color. When the MB dye test was performed
with Milli-Q water (Figure 8.7B), no bleaching or discoloration was observed. As shown
in Figure 8.7C, a MB dye test of 50/50 Milli-Q H20/ACN solvent produced diffuse
discoloration of the MB to light blue, spreading out/emanating fi'om the point of
application, with a dark blue band surrounding the discoloration. As shown in Figures
8.7D, E, and F, MB dye test strips tested with unquenched F enton’s reaction mixture at
15, 30, and 60 minutes, respectively, similarly indicated the presence of hydroxyl radicals
by an immediate concentrated white discoloration (bleaching) at the point of application
surrounded by diffuse discoloration of the MB to light blue, spreading out/emanating
from the point of application, with a dark blue band surrounding the discolored area.

After the specified remediation reaction period, each Fenton’s reaction was
quenched with 6 drops (approximately 0.24 mL) of 10% Na2803 solution. Immediately
following the addition of the 10% NaZSO3, the pH of the reaction mixture increased from
approximately 3 to 9. No significant change occurred in the temperature of the reaction

mixture. For each remediation, the addition of 10% NaZSO3 resulted in a change in

220

Figure 8.7 Fenton’s remediation of a solution of 4,4'-dichlorobiphenyl in 50/50 Milli-Q
H20/ACN. Methylene blue dye test results for (A) control (no sample added); (B) Milli-
Q water; (C) 50/50 Milli-Q H20/ACN solvent; unquenched Fenton’s reaction mixture at
(D) 15 minutes, (E) 30 minutes, and (F) 60 minutes; and Fenton’s reaction mixture
quenched with (G) 6 drops and (H) 8 drops of 10% Na2803. All methylene blue dye tests

were performed using 40 uL samples.

221

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reaction mixture appearance to clear and colorless with very fine rust colored (light
orange) particles in suspension. The appearance of rust colored particles can be
attributed to the more basic pH of the reaction mixture during the quenching process,
resulting in the conversion of iron from a hydrated ferrous form to a colloidal ferric form
and the formation of ferric hydroxide (10 -12). As shown in Figure 8.7G, the MB dye
test of the F enton’s reaction mixture quenched with 6 drops of 10% NaZSO3 solution
produced results that closely resembled the MB result indicated for 50/50 Milli-Q
H20/ACN solvent. The similarity of the MB dye test results for the quenched reaction
mixture and 50/50 Milli-Q H20/ACN solvent, as well as the absence of an immediate
concentrated white discoloration (bleaching) at the point of application, indicated that
quenching was complete and no hydroxyl radicals remained.

As a precautionary measure, an additional 2 more drops (approximately 0.09 mL)
of 10% Na2803 solution was added to each reaction mixture. Following the addition of
the additional 10% NaZSO3, the pH and appearance of the reaction mixture remained
unchanged from that observed following the initial addition of 10% NaZSO3. As shown
in Figure 8.7H, the MB dye test of the F enton’s reaction mixture quenched with an
additional 2 drops of 10% Na2803 solution (a total of 8 drops) produced results that
closely resembled the MB results indicated for 50/50 Milli-Q H20/ACN solvent and the
F enton’s reaction mixture quenched with 6 drops of 10% NaZSO3 solution, thereby
verifying that quenching was complete. For the MB dye test of the F enton’s reaction
mixture quenched with a total of 8 drops of 10% Na2803 solution, the slight white film
observed over the surface of the test area and spreading beyond the dark blue band

surrounding the area of diffuse discoloration can be attributed to excess 10% NaZSO3.

223

To aid in the removal of iron from the reaction mixture by precipitation of F e3 +,
following quenching, the reaction mixture was adjusted to pH 9 (unless the pH was
already greater than or equal to pH 9). Following this pH adjustment, the solution
remained clear and colorless with very fine rust colored (light orange) particles in
suspension. Since none of the reaction mixtures were highly concentrated with
precipitate, no centrifugation was performed prior to filtration. No rinsing of the
filtration funnel with Milli-Q water was necessary for the filtration of any of the reaction
mixtures. The filter paper was uniform light rust in color following filtration. The
filtrate appeared clear and colorless with no precipitate. Following adjustment of the
filtrate to pH 7, the solution remained clear and colorless with no precipitate.

Throughout each of the F enton’s remediation experiments, the amount of water
added by addition of 5.0 mM FeSO4, pH adjustment, addition of 3% H202, quenching,
and rinsing of the filtration funnel was monitored, and an adjusted total H20/ACN ratio
by volume was calculated at the completion of the experiment. The total H20/ACN ratio
suggested the degree of dilution of the sample that occurred during the experiment and
was maintained as constant as possible between the remediation experiments to allow for
comparison of the results. The original H20/ACN ratio at the start of the remediation
experiment, based on the solution used to dissolve the 4,4'DCBP, was 50/50 or 1.00. At
the completion of the experiments, the calculated adjusted total H20/ACN ratios for “0”,
15, 30, and 60 minutes of remediation were 1.07, 1.11, 1.12, and 1.11, respectively.
Since these H20/ACN ratios result in an average :1: standard deviation of 1.10 i 0.02, the

coefficient of variation is only 2.01%, which is within the allowable pipetting error for

224

dispensing liquids. This indicates that the ratio of H20/ACN did not significantly change
between the remediation experiments.

As described previously, two solutions of different expected “concentrations”
(156.1 and 78.05 ppm 4,4'DCBP/isooctane) were prepared by isooctane extraction from
each of the final F enton’s remediation solutions. Immediately following the extraction
process, the extraction mixture was a white emulsion. Following a rest period, a distinct
clear top and bottom layer partially separated by a white ring of froth, along the perimeter
of the extraction vial, were visible in the extraction mixture. The top layer dipped
through the center of the froth ring, but remained distinct fi'om the bottom layer. The top
extraction layer, containing 4,4'DCBP and isooctane, was carefully removed for GC/ECD

analysis.

8.3.4 GC/ECD Analysis and Disappearance of Parent PCB

The gas chromatogram for the blank (isooctane) revealed no significant peaks,
except for small peaks of impurities. From the gas chromatograms for the calibration
curve, the retention time (average :2 standard deviation) for 4,4’DCBP was determined to
be 15.20 i 0.04 minutes with a range of 15.16 to 15.27 minutes. Calibration curve gas
chromatograms for 40, 80, and 160 ppm 4,4’DCBP/isooctane are shown in Figures 8.8,
8.9, and 8.10, respectively. These chromatograms are representative of those observed
for the calibration curve determination. Two very small peaks with retention times of
approximately 8 and 12 minutes were also observed on the calibration curve gas
chromatograms; however, since these peaks were also observed on the gas chromatogram

for the blank (isooctane), they can be attributed to impurities in the isooctane. These

225

Figure 8.8 Representative calibration curve gas chromatogram for 40 ppm
4,4’DCBP/isooctane.

226

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impurity peaks were most evident on the gas chromatograms plotted with a more limited
scale on the response (vertical) axis, such as Figure 8.8. The equation determined by
linear regression for the calibration curve (Figure 8.11) was y = 17018x — 58658 with an
R2 = 0.9969.

The gas chromatograms for the extracted Fenton’s remediation solutions indicated
only a single major peak, the parent PCB, 4,4'DCBP, with a retention time (average i
standard deviation) of 15.24 i 0.05 minutes with a range of 15.17 to 15.31 minutes. This
retention time correlates closely with that found for the 4,4'DCBP/isooctane standards
used for the calibration curve. Gas chromatograms for the “78.05 ppm” and “156.1 ppm”
4,4'DCBP/isooctane extraction samples from the Fenton’s remediation solution reacted
for 60 minutes are shown in Figures 8.12 and 8.13, respectively. These chromatograms
are representative of those observed for the extraction sample “concentrations” prepared
from F enton’s remediation solutions reacted for “0”, 15, 30, and 60 minutes. As
discussed previously, two very small peaks with retention times of approximately 8 and
12 minutes were also observed on the gas chromatograms from the extraction samples,
but are attributable to impurities in the isooctane.

No remediation byproduct peaks were observed for any of the extraction samples
from the Fenton’s remediation solutions. The peaks observed for the potential
remediation byproducts, 3-chloro-2-biphenylol (retention time 14.21 minutes) and 4,4'-
dichloro-3-biphenylol (retention time 17.03 minutes), were also not observed. One
possible explanation for the absence of remediation byproduct peaks might be the
production of remediation byproducts in concentrations too small to be detected with the

ECD. The ECD is more sensitive to detecting highly chlorinated compounds. If a

232

Figure 8.11 Calibration curve for 4,4'DCBP/isooctane.

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Figure 8.12 Gas chromatogram for the “7 8.05 ppm” 4,4'DCBP/isooctane extraction
sample fi'om the Fenton’s remediation solution reacted for 60 minutes (pH 3.0,
temperature 23.0 °C, Fe2+zH202 molar ratio 1:20, initial Fe2+ concentration 0.15 mM, and
initial H202 concentration 3 mM).

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Time (minutes)

remediation byproduct contains only a few chlorines, a higher concentration might be
required for the ECD to detect it. Since the parent compound, 4,4'DCBP, contains only
two chlorines, remediation might have resulted in byproducts that are not easily
detectable by ”the ECD. Absence of remediation byproduct peaks might also be attributed
to further remediation of remediation byproducts, resulting in the production of
secondary and tertiary byproducts. Instead of large concentrations of primary
byproducts, further remediation could result in numerous compounds in smaller
concentrations, possibly below detectable levels. Finally, the absence of remediation
byproduct peaks might be a consequence of the generation of dechlorinated byproducts,
which are undetectable by ECD.

For “0 minutes” of remediation, the concentration of the final Fenton’s
remediation solution (average i standard deviation) was determined to be 150.77 i 10.44
ppm 4,4’DCBP, which is equivalent to removal of 66.19% of the original 446 ppm
4,4'DCBP (33.81% 4,4'DCBP remaining). Since “0 minutes” of remediation was defined
as the remediation procedure just prior to the initiation of the Fenton’s reaction by the
addition of 3% H202, no reaction should have occurred and the concentration should have
remained at 446 ppm 4,4'DCBP. The results of the Fenton’s remediation of 4,4'DCBP
indicated that initiation of the F enton’s reaction by the addition of 3% H202 was
necessary to solubilize the 4,4 'DCBP. Despite repeated sonication, prior to the initiation
of the Fenton’s reaction the reaction mixture was clear and colorless with very fine white
particles in suspension. Following initiation of the Fenton’s reaction, the amount of very
fine white particles in suspension decreased as the reaction progressed, until no

precipitate was apparent, after about 5 minutes. Since there was no initiation of the

239

Fenton’s reaction for “0 minutes” of remediation, any undissolved 4,4'DCBP would have
been removed from the solution during the filtration process. Hence, the removal of
4,4’DCBP during the “0 minutes” of remediation experiment can be attributed to loss via
the filtration process. Since it is impossible to obtain a true zero time of remediation, as
this would require instantaneously quenching the reaction following its initiation, and
complete dissolution of 4,4'DCBP requires initiation of the reaction, the decision was
made to consider the original concentration of 4,4’DCBP (446 ppm) to be the 0 minutes
of remediation concentration. The possibility of losses of 4,4 ’DCBP during the filtration
process for the 15, 30, and 60 minutes of remediation experiments were not evaluated;
however, these could constitute potential further studies.

As described previously, the concentrations of the final Fenton’s remediation
solutions were determined through a process of back-calculation from the GC/ECD
results. Since these calculations assumed 100% isooctane extraction efficiency, the
actual percent isooctane extraction efficiency was determined fi'om the percent of ACN in
the dilution water and the results of the “Percent Efficiency of Recovery by Isooctane
Extraction” experiment. The percents of ACN in the dilution water for the “156.1 ppm”
and “78.05 ppm” 4,4’DCBP/isooctane extraction samples were 0.57% and 0.29%,
respectively. From the “Percent Efficiency of Recovery by Isooctane Extraction”
experiment, a mathematical relationship was developed between the percent of ACN in
the dilution water and the isooqtane extraction efficiency. By applying linear regression
to the results corresponding to 0.33% and 0.66% ACN, which are closest to the percent of
ACN of interest for the 4,4'DCBP/isooctane extraction samples, the equation y = 100.33

— 12.45x with an R2 = 1.00 was determined where “x” is the percent of ACN an “y” is

240

the percent efficiency of isooctane extraction. By applying this equation to the percent of
ACN in the dilution water for the “156.1 ppm” and “78.05 ppm” 4,4'DCBP/isooctane
extraction samples, the actual percent isooctane extraction efficiencies were calculated to
be 93.23% and 96.72%, respectively. Since both actual percent isooctane extraction
efficiencies were greater than ninety percent, no mathematical adjustment of the final
Fenton’s remediation solution concentrations of 4,4'DCBP was required.

Figure 8.14 presents a graph of the average percent of 4,4'DCBP remaining in the
final Fenton’s remediation solution versus the remediation time. The original
concentration of 4,4'DCBP (446 ppm) was assumed to be the 0 minutes of remediation
concentration, corresponding to 100% for an average percent of 4,4'DCBP remaining.
By 15 minutes of remediation, a decrease in the average concentration of the final
Fenton’s remediation solution occurred, resulting in 27.22 i 1.32% (average i standard
deviation) of 4,4'DCBP remaining (equivalent to a removal of 72.78 :I: 1.32% of the
4,4'DCBP). No further significant decrease in the average concentration of the final
Fenton’s remediation solution occurred with further remediation up to 60 minutes. Using
Kruskal-Wallis One Way Analysis of Variance on Ranks, there was no statistically
significant difference between the average percents of 4,4 'DCBP remaining in the final
Fenton’s remediation solution remediated for 15, 30, and 60 minutes. It is unlikely that
removal of the 4,4'DCBP is a result of sorption onto the reaction vessel walls, since the
amount of 4,4'DCBP is small compared to the large surface area of the reaction vessel
walls, which would have resulted in almost complete removal of the 4,4'DCBP.
Furthermore, according to Sedlak et al. (13), the rapid kinetics of the hydroxyl reactions

with PCBs relative to adsorption to container walls assures that the reaction of hydroxyl

241

Figure 8.14 Graph of the average percent of 4,4'—dichlorobiphenyl remaining following
Fenton’s remediation vs. the remediation time.

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radicals with PCBs would be complete before any PCBs were adsorbed to the container
walls.

The incomplete removal of 4,4'DCBP can be attributed to multiple factors. One
explanation for the incomplete removal observed for the Fenton’s remediation of
4,4’DCBP is that the H202 was the limiting compound. The H202 could have been
depleted during the remediation of the 4,4'DCBP, so that no further removal of 4,4'DCBP
occurred after that observed at 15 minutes. Similar results were observed by Trapido et
al. (7), where incomplete degradation of nitrophenols by Fenton’s reagent occurred for a
variety of Fe2+zH202 ratios, including 1:20. H202 was concluded to be the limiting
compound causing the remediation of nitrophenols to become restrained such that no
further degradation took place. Likewise, experiments performed by Kuo et al., (14)
involving the remediation of aqueous 4-chlorobiphenyl and 4,4'-dichlorobiphenyl by
Fenton’s reaction, resulted in a constant concentration of chlorobiphenyls indicative of
incomplete degradation.

As future studies, a variety of experiments could be performed to verify that H202
is the limiting compound responsible for the incomplete removal of 4,4 'DCBP under the
conditions investigated. One experiment could involve increasing the initial
concentration of H202 to a value greater than 3 mM while keeping the initial Fe2+
concentration at 0.15 mM. Alternatively, the F enton’s reaction could be conducted under
the same conditions previously investigated; however, additional H202 could be added to
the reaction mixture after an initial reaction period of 15 minutes. In either experiment,
greater removal of 4,4'DCBP than previously observed would indicate that H202 is the

limiting compound. An alternative experiment would be to measure the residual

244

concentration of H202 after varying Fenton’s remediation times. If the H202 has been
depleted when no further removal of 4,4'DCBP occurs, H202 will be verified as the
limiting compound. One procedure for measuring the residual concentration of H202 is
the photometric method for the determination of low concentrations of H202 by the
peroxidase catalyzed oxidation of N,N-diethyl-p-phenylenediamine (DPD) described by
Bader et a1. (15)

Although incomplete removal of 4,4'DCBP occurred, hydroxyl radicals were still
observed by the MB dye test through 60 minutes of Fenton’s remediation. Despite the
apparent depletion of H202, hydroxyl radicals might have continued to be generated
(although perhaps at a lower concentration) because of the persisting radical chain
reaction. Since the MB dye test only qualitatively detects the presence of hydroxyl
radicals, it is not possible to determine the quantity of hydroxyl radicals present at each of
the F enton’s remediation times. Preferential reaction of the hydroxyl radicals with the

byproducts rather than the 4,4'DCBP would result in no further removal of 4,4’DCBP.

8.4 Conclusions

Fenton’s remediation of 4,4'DCBP in 50/50 Milli-Q H20/ACN was performed for
0, 15, 30, and 60 minutes. To prepare the remediation mixtures for GC/ECD analysis, an
isooctane liquid-liquid extraction method was developed that resulted in greater than
ninety percent efficiency of recovery of 4,4'DCBP when the percent of ACN in the
dilution water during the extraction procedure was less than 0.66%. The utilization of
Milli-Q H20 as a means of diluting the ACN in the extraction process was essential to

obtaining an acceptable efficiency of recovery of 4,4'DCBP by isooctane extraction. The

245

gas chromatograms for the extracted F enton’s remediation solutions indicated only a
single major peak, the parent PCB, 4,4'DCBP, with a retention time (average i standard
deviation) of 15.24 :t 0.05 minutes. No remediation byproduct peaks or the peaks
observed for the potential remediation byproducts, 3-chloro-2-biphenylol (retention time
14.21 minutes) and 4,4'-dichloro-3-biphenylol (retention time 17.03 minutes), were
observed for any of the extraction samples from the Fenton’s remediation solutions.
Since complete dissolution of 4,4'DCBP required initiation of the reaction, the original
concentration of 4,4'DCBP (446 ppm) was assumed to be the 0 minutes of remediation
concentration. A decrease in the average concentration of the final Fenton’s remediation
solution occurred by 15 minutes of remediation, resulting in 27 .22 i 1.32% (average i
standard deviation) of 4,4'DCBP remaining (equivalent to a removal of 72.78 :t 1.32% of
the 4,4'DCBP). No firrther significant decrease in the average concentration of the final

Fenton’s remediation solution occurred with further remediation up to 60 minutes.

246

8.5 References

1. Hemer, H.A.; Trosko, J.E.; Masten, 8.]. The Epigenetic Toxicity of Pyrene and
Related Ozonation Byproducts Containing an Aldehyde Functional Group. Environ.
Sci. Technol. 2001, 35 (17), 3576-3583.

2. Gankin, Y.V.; Gorshteyn, A.E.; Robbat, A., Jr. Identification of PCB Congeners by
Gas Chromatography Electron Capture Detection Employing a Quantitative
Structure-Retention Model. Anal. Chem. 1995, 67 (15), 2548-2555.

3. Method 8082A: Polychlorinated Biphenyls (PCBs) by Gas Chromatography. Test
Methods for Evaluating Solid Waste, Physical/Chemical Methods; SW-846; US.
Environmental Protection Agency, US. Government Printing Office: Washington,
DC, 2000.

4. Skoog, D.A.; Holler, F.J.; Neirnan, T.A. Principles of Instrumental Analysis, 5th ed.;
Harcourt Brace & Company: Orlando, FL, 1998; pp 708-709.

5. Micaroni, R.C.C.M.; Bueno, M.I.M.S.; Jardim, W.F. Degradation of Acetonitrile
Residues Using Oxidation Processes. J. Braz. Chem. Soc. 2004, 15 (4), 509-513.

6. Satoh, A.Y.; Trosko, J.E.; Masten, S.J. Epigenetic Toxicity of Hydroxylated
Biphenyls and Hydroxylated Polychlorinated Biphenyls on Normal Rat Liver
Epithelial Cells. Environ. Sci. T echnol. 2003, 3 7 (12), 2727-2733.

7. Trapido, M; Goi, A. Degradation of nitrophenols with the Fenton reagent. Proc. Est.
Acad Sci. Chem. 1999, 48(4), 163-173.

8. Mohamadin, A.M. Possible role of hydroxyl in the oxidation of dichloroacetonitrile
by Fenton-like reaction. J. Inorg. Biochem. 2001, 84 (1-2), 97-105.

9. Trapido, M. Tallinn Technical University, Tallinn, Estonia. Personal Communication,
2002.

10. Lindsey, M. E.; Tarr, M. A. Quantitation of hydroxyl radical during Fenton oxidation
following a single addition of iron and peroxide. Chemosphere 2000, 41 (3), 409-417.

11. Arnold, S. M.; Hickey, W. J .; Harris, R. F. Degradation of atrazine by Fenton’s
reagent: condition optimization and product quantification. Environ. Sci. Technol.

1995, 29(8), 2083-2089.

12. Pratap, K.; Lemley, A. T. F enton electrochemical treatment of aqueous atrazine and
metolachlor. J. Agric. Food Chem. 1998, 46(8), 3285-3291.

13. Sedlak, D.L.; Andren, A.W. Aqueous-Phase Oxidation of Polychlorinated Biphenyls
by Hydroxyl Radicals. Environ. Sci. Technol. 1991, 25 (8), 1419-1427.

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14. Kuo, C.-Y.; Lo, S.-L. Oxidation of aqueous chlorobiphenyls with photo-Fenton
process. Chemosphere 1999, 38 (9), 2041-2051.

15. Bader, H.; Sturzenegger, V.; Hoigné, J. Photometric method for the determination of
low concentrations of hydrogen peroxide by the peroxidase catalyzed oxidation of
N,N-diethyl-p-phenylenediamine (DPD). Water Res. 1988, 22 (9), 1109-1115.

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Chapter 9

Summary and Recommendations

9.1 Summary

The primary goal of this research was to perform F enton’s remediation of 4,4'-
dichlorobiphenyl (4,4'DCBP) under a set of selected conditions, followed by an
examination of the toxicity of the final F enton’s remediation solution and disappearance
of 4,4'DCBP. The first division of research involved toxicology studies of potential
Fenton’s remediation byproducts and the selected parent PCB (4,4'-dichlorobiphenyl).
Six commercially available chemicals considered to be representative of OH-BPs
(hydroxylated biphenyls) and OH-PCBs (hydroxylated polychlorinated biphenyls), which
are potential byproducts of remediation of PCBs with Fenton’s reagent, were selected for
study of the correlation between their chemical properties (including structure) and
observed epigenetic toxicity and cytotoxicity. These six chemicals were 2-biphenylol
(2BP), 3-biphenylol (3 BP), 2,2’-biphenyldiol (2,2'BP), 3,3'-biphenyldiol (3,3'BP), 3-
chloro-2-biphenylol (3C2BP), and 4,4'—dichloro-3-biphenylol (4,4'DC3BP). Only 3,3'-
biphenyldiol and 4,4'-dichloro-3-biphenylol induced cytotoxicity within a dose range of 0
to 300 uM. Noncytotoxic doses were selected for evaluation of epigenetic toxicity. 4,4'-
Dichloro-3 -biphenylol was most inhibitory to GJIC at the lowest dose. 3-Chloro-2-
biphenylol was least inhibitory to GJIC. 3-Chloro-2-biphenylol was less inhibitory to
GJIC than 2-biphenylol because of the presence of the chlorine functional group, which

appears to attenuate the toxic effect of the ortho-hydroxyl group. Although cells were

capable of complete recovery of GJIC after removal of each of the chemicals, only with

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2,2'-biphenyldiol and 4,4'—dichloro-3-biphenylol did the cells demonstrate partial
recovery without the removal of the chemical. The dose-response results for the selected
parent PCB, 4,4'DCBP, were consistent with observations that PCBs with coplanar
conformations appear less likely to inhibit GJIC. For a dose range of 0 to 260.87 uM,
very slight to no inhibition of GJIC was observed for 4,4'DCBP for the dose-response
experiments for incubation times of 30 minutes, 2 hours, 6 hours, and 24 hours.
Although 4,4’DCBP was not observed to be significantly inhibitory to GJIC, Fenton’s
remediation of this chemical has the potential of generating byproducts which are
inhibitory to GJIC and/or cytotoxic (based on the potential of forming byproducts such as
4,4'DC3BP).

In the second division of research, the methylene blue (MB) dye test was
developed as a new test that qualitatively indicates the presence of hydroxyl radicals
through an immediate, distinct bleaching of the MB dye on a paper test strip after
applying an aqueous sample containing hydroxyl radicals. When applied to Fenton’s
remediation, MB dye tests were capable of indicating the formation of hydroxyl radicals
during the F enton’s reaction and indicating the completion of quenching (absence of
hydroxyl radicals) during the quenching process. The presence of hydroxyl radicals was
verified by benzoic acid chemical probe hydroxyl radical detection methods using thin
layer chromatography (TLC) and spectrophotometric wavelength scans. The presence of
hydroxyl radicals was indirectly determined by the detection of hydroxylated benzoic
acids on TLC plates and a violet colored solution with a peak absorbance at a wavelength

close to 520 nm. In experiments to test the performance of the methylene blue dye test

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strips, the age of the test strip, the presence of H202, and sample pH were determined to
have no significant effect on the methylene blue dye test results.

In the third division of research, it was verified that the reaction of F enton’s
reagent with the solvents (Milli-Q H20 alone, 80/20 Milli-Q H20/ACN (by volume), and
50/50 Milli-Q H20/ACN (by volume)) in the absence of 4,4'DCBP, does not result in any
toxic byproducts. For the final reaction mixture solutions of F enton’s remediation in
Milli-Q H20 alone, corresponding to the Fe2+zH202 ratios 1:5, 1:20, and 1:40, no
inhibition of GJIC was observed for the dose-response GJIC bioassay for incubation
times of 30 minutes (0 to 300 1.1L) and 2 hours (300 uL). Hence, F enton’s reagent
remediation with water alone is not expected to result in toxicity, and any toxicity
resulting from the Fenton’s reagent remediation of 4,4'DCBP can be assumed to be
independent of the influence of water. The remediation procedure developed in this
series of experiments was later applied to the F enton’s remediation of 4,4'DCBP. Based
on the results of Fenton’s remediation in Milli-Q water, an F e2+zH202 ratio of 1:20 was
selected for the F enton’s remediation of 4,4’DCBP, since it appeared to result in greater
production of hydroxyl radicals than 1:40 and resulted in less F e3 + precipitate prior to
filtration than that observed with the ratio 1 :5.

Fenton’s remediation of the solvents 80/20 Milli-Q H20/ACN and 50/50 Milli-Q
H20/ACN (by volume) were investigated to determine the toxicological effect of ACN in
combination with water as a solvent in remediation. In addition, the application of the
MB dye test for Fenton’s reaction in Milli-Q H20/ACN was examined. Although neither
Milli-Q water nor 100% ACN alone resulted in bleaching or discoloration of the MB,

Milli-Q H20/ACN solvent resulted in the discoloration of the MB unique to the 80/20

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and 50/50 volume ratios examined. As was observed for Milli-Q H20 with varying pH
values, the MB dye test results for 80/20 Milli-Q H20/ACN solvent were not influenced
by sample pH. Since hydroxyl radicals appeared to be produced throughout the reaction
period, the ACN does not appear to have a scavenging effect on the hydroxyl radicals to
the extent detectable by the MB dye test. For each of the final reaction mixture solutions
from Fenton’s remediation of 80/20 Milli-Q H20/ACN and 50/50 Milli-Q H20/ACN
solvents, no inhibition of GJIC was observed with 30 minutes of incubation time for
volume ranges of 0 to 150 uL and 0 to 60 p.L, respectively. Furthermore, for the highest

tested volumes of the final reaction mixture solutions, no inhibition of GJIC was

 

observed at 2 hours of incubation. Hence, F enton’s remediation of 80/20 Milli-Q
H20/ACN and 5 0/50 Milli-Q H20/ACN solvents alone did not result in any toxicity, and
any toxicity resulting from the Fenton’s remediation of 4,4'DCBP in either of these

solvents can be assumed to be independent of the influence of these solvents. The

remediation procedure further developed in this series of experiments was later applied to
the Fenton’s remediation of 4,4'DCBP.

The fourth division, involved Fenton’s remediation of 4,4'DCBP in 50/50 Milli-Q
H20/ACN, followed by the examination of the toxicity of the final Fenton’s remediation
solution and disappearance of 4,4'DCBP as a result of Fenton’s remediation. The
Fenton’s reaction conditions were pH 3.0, temperature 23.0 °C, Fe2+zH202 molar ratio
1:20, initial Fe2+ concentration 0.15 mM, and initial H202 concentration 3 mM. The pre-
remediation concentration of 4,4'DCBP in the reaction mixture was 2 mM (or 446 ppm).
The quenched and unquenched MB dye test results for Fenton’s remediation of a solution

of 4,4'DCBP in 50/50 Milli-Q H20/ACN were identical in appearance to those observed

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for F enton’s remediation of 50/50 Milli-Q H20/ACN solvent. It can therefore be
concluded that the ability of the MB dye test to indicate the presence of hydroxyl radicals
does not appear to be influenced by the presence of 4,4’DCBP.

For the 30 minute dose-response GJIC bioassay of the final Fenton’s remediation
solution, a gradual decline in GJIC was observed with an increase in test volume. Partial
inhibition of GJIC occurred for test volumes greater than 40 uL, and a maximum level of
inhibition was attained at 60 uL (FOC = 0.75 d: 0.03). For the time-response GJIC
bioassay of the final Fenton’s remediation solution (60 uL test volume) a maximum level
of inhibition was attained at 30 minutes (F CC = 0.78 i 0.02) of chemical exposure
followed by complete recovery of GJIC without removal of the chemical by 240 minutes
of chemical exposure. The complete recovery of GJIC without removal of the chemical
observed for the final F enton’s remediation solution was similar to the partial recovery of
GJIC without removal of the chemical observed for the potential remediation byproduct
4,4'-dichloro-3 -biphenylol (4,4’DC3BP). The inhibitory effects of 4,4'DC3BP previously
observed might have been attenuated by its presence at a lower concentration as a
component of the final Fenton’s remediation solution. The final F enton’s remediation
solution was determined to be more toxic (inhibitory to GJIC) than the parent PCB,
4,4’DCBP, which exhibited very slight to no inhibition of GJIC for the dose-response
experiments for incubation times of 30 minutes, 2 hours, 6 hours, and 24 hours.

Fenton’s remediation of 4,4'DCBP in 50/50 Milli-Q H20/ACN was performed for '
0, 15, 30, and 60 minutes. Isooctane extracted Fenton’s remediation solution samples

were analyzed by GC/ECD to quantitate the disappearance of the parent PCB, 4,4'DCBP,

over the period of remediation. The gas chromatograms for the extracted Fenton’ s

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remediation solutions indicated only a single major peak, the parent PCB, 4,4'DCBP,
with a retention time (average i standard deviation) of 15.24 :1: 0.05 minutes. No
remediation byproduct peaks or the peaks observed for the chlorinated potential
remediation byproducts, 3-chloro-2-biphenylol (retention time 14.21 minutes) and 4,4'-
dichloro-3-biphenylol (retention time 17.03 minutes), were observed for any of the
extraction samples from the Fenton’s remediation solutions. Although the final Fenton’s
remediation solution was determined to be more toxic (inhibitory to GJIC) than the l
parent PCB and the inhibitory effects were similar to those observed for the potential 1

remediation byproduct 4,4'DC3BP, the byproducts responsible for the toxicity observed

 

could not be ascertained by the detection methods employed. A decrease in the average
concentration of the final Fenton’s remediation solution occurred by 15 minutes of
remediation, resulting in 27.22 i- 1.32% (average i stande deviation) of 4,4’DCBP
remaining (equivalent to a removal of 72.78 i 1.32% of the 4,4'DCBP). No further

significant decrease in the average concentration of the final Fenton’s remediation

solution occurred with further remediation up to 60 minutes.

9.2 Recommendations

In view of the results obtained in this research, a series of possible further studies
might be conducted. For the Fenton’s remediation of 4,4'DCBP, a decrease in the
average concentration of 4,4'DCBP in the final Fenton’s remediation solution occurred
by 15 minutes of remediation with no further decrease with firrther remediation up to 60
minutes. One explanation for the incomplete removal observed for the Fenton’s

remediation of 4,4'DCBP is that the H202 was the limiting compound. The H202 could

254

have been depleted during the remediation of the 4,4'DCBP, so that no further removal of
4,4’DCBP occurred after that observed at 15 minutes. In future studies, a variety of
experiments could be performed to verify that H202 is the limiting compound responsible
for the incomplete removal of 4,4'DCBP under the conditions investigated. One
experiment could involve increasing the initial concentration of H202 to a value greater
than 3 mM while keeping the initial F e2+ concentration at 0.15 mM. Alternatively, the
Fenton’s reaction could be conducted under the same conditions previously investigated;
however, additional H202 could be added to the reaction mixture after an initial reaction
period of 15 minutes. In either experiment, greater removal of 4,4'DCBP than previously
observed would indicate that H202 is the limiting compound. An alternative experiment
would be to measure the residual concentration of H202 after varying Fenton’s
remediation times. If the H202 has been depleted when no further removal of 4,4'DCBP
occurs, H202 will be verified as the limiting compound. One procedure for measuring the
residual concentration of H202 is the photometric method for the determination of low
concentrations of H202 by the peroxidase catalyzed oxidation of N,N-diethyl-p-
phenylenediamine (DPD).

For the F enton’s remediation of 4,4’DCBP, since the byproducts responsible for
the toxicity observed for the final F enton’s remediation solution could not be ascertained
by the detection methods employed, despite a decrease in the average concentration of
4,4 ’DCBP in the final F enton’s remediation solution, alternative methods could be
performed to detect the byproducts. The byproducts might be detectable by gas
chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass

spectrometry (LC/MS). An alternative series of experiments could involve fractionation

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of the final Fenton’s remediation solution by high-performance liquid chromatography
(HPLC) followed by identification of the fractions by nuclear magnetic resonance (NMR)
and GC/MS. In addition, toxicology studies could be performed on the fiactions to
identify which compounds are associated with the toxicity of the final Fenton’s
remediation solution. These results could also be compared to the toxicology and
structures of the six selected potential remediation byproducts, the parent PCB, and other

OH-PCB/OH-BP standards.

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